Recombinant cytomegalovirus vectors as vaccines for tuberculosis

ABSTRACT

The present disclosure provides cytomegalovirus vectors encoding fusion proteins comprising  Mycobacterium tuberculosis  (Mtb) antigens, nucleic acid molecules encoding the same, cytomegalovirus vectors comprising nucleic acid molecules, compositions comprising the same, and methods of eliciting an immune response against tuberculosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 62/353,432 filed Jun. 22, 2016 and U.S. Provisional Application Ser.No. 62/478,099 filed Mar. 29, 2017, each of which is incorporated hereinby reference in its entirety.

FIELD

The present disclosure is directed, in part, to cytomegalovirus vectorsencoding fusion proteins comprising Mycobacterium tuberculosis (Mtb)antigens, nucleic acid molecules encoding the same, cytomegalovirusvectors comprising nucleic acid molecules, compositions comprising thesame, and methods of eliciting an immune response against tuberculosis.

BACKGROUND

Tuberculosis (TB) is a global health problem resulting in 8 million newcases and 2 million deaths each year. The emergence of multi-drug andtotally-drug resistant strains of TB only makes this problem moresevere. The life cycle of Mtb has 3 stages. In the acute phase followinginitial infection the bacteria replicate in the host and virulencefactors are expressed, leading to the generation of an immune responseby the host. As the immune response begins to control the infection, theMtb enters a latent, asymptomatic state in which the bacteria becomenon-replicating and are encased in granulomas. The bacterium can persistin this latent state in infected individuals for many years, makingdiagnosis and treatment of disease difficult. In some cases, thebacteria are reactivated and begin replicating again, leading back tothe disease state. Reactivation can occur for numerous reasons,including immune suppression caused by diseases such as HIV, treatmentssuch as chemotherapy, or the weakening of the immune system due toaging. An estimated 2 billion people are latently infected with Mtbworldwide, and reactivation of latent Mtb accounts for most new cases ofactive TB disease. Reactivation is associated with inflammation,necrosis and cavitation of the lung, a process that results in drainingof the lesions into the bronchus. Aerosols generated when individualswith bronchial lesions cough causes dissemination of the Mtb organism touninfected, susceptible persons, and the transmission cycle is thusmaintained.

The only currently available vaccine against TB, Mycobacterium bovis(Bacille Calmette-Guérin) (BCG), was first introduced in 1921. BCG hasbeen widely utilized and while studies show that for some purposes BCGis effective (e.g. against disseminated TB in infants), it is known tobe ineffective with respect to preventing the development, persistenceand reactivation of latent TB in adults. There is an ongoing need todevelop improved, more effective vaccines against TB.

Use of cytomegalovirus (CMV) vectors (e.g., Rhesus CMV (RhCMV) and humanCMV (HCMV)) has particular advantages. First, CMV elicits anastoundingly high frequency (steady-state) T cell response, at least anorder of magnitude higher than that of most non-persistent virus (it isnot uncommon for CMV-specific T cells to encompass >20% of thecirculating memory repertoire), and the representation of CMV-specific Tcells (as it relates to CMV-driven non-CMV antigens) is even higher intissues such as the lung and liver. In addition, the above responsespersist indefinitely. CMV is also capable of re-infecting alreadychronically infected individuals, even in the face of pre-existingimmune responses, and such re-infection with recombinant CMVs is alsocapable of inducing new responses to distinct CMV-encoded foreignproteins. CMV also engenders pathogenicity only in very specificsituations of immune deficiency, immaturity, or seronegative pregnantwomen (its potential for disease is among the best documented amongpotential human pathogens). Finally, CMV infection is ubiquitous in mostof humanity.

While vaccines are often effective to immunize individualsprophylactically or therapeutically against pathogen infection or humandiseases, there is a need for improved vaccines and vectors. There isalso a need for compositions and methods that produce an enhanced immuneresponse. Likewise, while some immunotherapeutics are useful to modulateimmune response in a patient, there remains a need for improvedimmunotherapeutic compositions and methods.

SUMMARY

The present disclosure provides recombinant RhCMV or HCMV vectorscomprising a nucleic acid sequence encoding an expressible Mtb antigenselected from Ag85A-Ag85B-Rv3407, Rv1733-Rv2626c, RpfA-RpfC-RpfD,Ag85B-ESAT6, and Ag85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.

The present disclosure also provides pharmaceutical compositionscomprising the recombinant RhCMV or HCMV vaccine vectors describedherein and a pharmaceutically acceptable carrier.

The present disclosure also provides methods for treatment or preventionof tuberculosis comprising administering to a subject in need thereof atleast one recombinant RhCMV or HCMV vaccine vector described herein.

The present disclosure also provides methods for eliciting an immuneresponse to a Mtb antigen comprising administering to a subject in needthereof at least one recombinant RhCMV or HCMV vaccine vector describedherein.

The present disclosure also provides methods for eliciting a CD8+ orCD4+ T cell response to a Mtb antigen comprising administering to asubject in need thereof at least one recombinant RhCMV or HCMV vaccinevector described herein.

The present disclosure also provides Mtb antigens selected fromAg85B-ESAT6 and Ag85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.

A joint research agreement exists between Aeras and the Oregon Health &Sciences University.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows immunogenicity of BCG and Rhesus CMV vectors containingvarious TB constructs; immune responses induced by vaccination wereanalyzed by intracellular cytokine staining throughout the vaccinationperiod; shown is the percentage of memory cells expressing either IFNγor TNF; CD4+ T cells are shown in the upper panel and CD8+ T cells areshown in the lower panels.

FIG. 2 (panels a, b, c, and d) shows ESAT-6-specific responses analyzedby intracellular cytokine staining throughout the vaccination period;shown above are the percentages of memory cells expressing either IFNγor TNF; included are responses from peripheral blood mononuclear cells(PBMCs; shown in panels a and b) and bronchoalevolar lavage cells (BAL;shown in panels c and d); CD4+ T cells are shown in panels a and c, andCD8+ T cells are shown in panels b and d.

FIG. 3 (panels a, b, c, and d) shows Rv1733-specific responses analyzedby intracellular cytokine staining throughout the vaccination period;shown above are the percentages of memory cells expressing either IFNγor TNF; included are responses from peripheral blood mononuclear cells(PBMCs; shown in panels a and b) and bronchoalevolar lavage cells (BAL;shown in panels c and d); CD4+ T cells are shown in panels a and c, andCD8+ T cells are shown in panels b and d.

FIG. 4 (panels a, b, c, and d) shows RpfC-specific responses analyzed byintracellular cytokine staining throughout the vaccination period; shownabove are the percentages of memory cells expressing either IFNγ or TNF;included are responses from peripheral blood mononuclear cells (PBMCs;shown in panels a and b) and bronchoalevolar lavage cells (BAL; shown inpanels c and d); CD4+ T cells are shown in panels a and c, and CD8+ Tcells are shown in panels b and d.

FIG. 5 (panels a, b, c, and d) shows Ag85B-specific responses analyzedby intracellular cytokine staining throughout the vaccination period;shown above are the percentages of memory cells expressing either IFNγor TNF; included are responses from peripheral blood mononuclear cells(PBMCs; shown in panels a and b) and bronchoalevolar lavage cells (BAL;shown in panels c and d); CD4+ T cells are shown in panels a and c, andCD8+ T cells are shown in panels b and d.

FIG. 6 (panels a and b) shows Ag85A-specific responses analyzed byintracellular cytokine staining throughout the vaccination period; shownabove are the percentages of memory cells expressing either IFNγ or TNF;included are responses from peripheral blood mononuclear cells; CD4+ Tcells are shown in panel a and CD8+ T cells are shown in panel b.

FIG. 7 (panels a and b) shows Rv3407-specific responses analyzed byintracellular cytokine staining throughout the vaccination period; shownabove are the percentages of memory cells expressing either IFNγ or TNF;included are responses from peripheral blood mononuclear cells; CD4+ Tcells are shown in panel a and CD8+ T cells are shown in panel b.

FIG. 8 (panels a and b) shows Rv2626-specific responses analyzed byintracellular cytokine staining throughout the vaccination period; shownabove are the percentages of memory cells expressing either IFNγ or TNF;included are responses from peripheral blood mononuclear cells; CD4+ Tcells are shown in panel a and CD8+ T cells are shown in panel b.

FIG. 9 (panels a and b) shows Rpm-specific responses analyzed byintracellular cytokine staining throughout the vaccination period; shownabove are the percentages of memory cells expressing either IFNγ or TNF;included are responses from peripheral blood mononuclear cells; CD4+ Tcells are shown in panel a and CD8+ T cells are shown in panel b.

FIG. 10 (panels a and b) shows RpfA-specific responses analyzed byintracellular cytokine staining throughout the vaccination period; shownabove are the percentages of memory cells expressing either IFNγ or TNF;included are responses from peripheral blood mononuclear cells; CD4+ Tcells are shown in panel a and CD8+ T cells are shown in panel b.

FIG. 11 (panels a and b) shows Ag85B-specific cells phenotyped by flowcytometry and classified as naive, Tcm (central memory), TrEM(transitional effector memory), or Tem (effector memory) T cells; CD4+ Tcells are shown in panel a and CD8+ T cells are shown in panel b;analysis was performed in the plateau phase, at days 316/318 after BCGvaccination.

FIG. 12 shows T cells stimulated with antigen in the presence ofantibody to block MHC I (red) or MHC II (blue); CD8+ T cell responsesinduced by BCG are primarily inhibited by blocking MHC I; in contrast,responses induced by RhCMV 68-1 are primarily inhibited by blocking MHCII.

FIG. 13 shows correlation between various pairings of efficacy criteria;also included in each analysis are slope (rs) and p value.

FIG. 14 shows representative CT scans from NHP in the unvaccinated andRhCMV/TB groups.

FIG. 15 shows the volume of parenchymal disease present at the timepoints indicated (or, for data points shown with open symbols, atnecropsy).

FIG. 16 (panels a, b, and c) shows the necropsy score overall (panel a),in the lung alone (panel b) and in lung draining lymph nodes (panel c).

FIG. 17 (panels a, b, c, and d) shows the bacterial burden (CFU/gtissue) present in random samples from the lung/trachea (light blue),draining lymph nodes (red), peripheral lymph nodes (dark blue), andextrapulmonary tissues (green); shown are representative NHP from eachgroup, including unvaccinated (panel a), BCG (panel b), CMV (panel c),and BCG+CMV (panel d).

FIG. 18 shows the number of Mtb culture-positive samples present inrandom biopsies from NHP; overall culture scores are broken out intolung and non-lung compartments.

FIG. 19 (panels a and b) shows non-lung samples further broken out intolung draining lymph node samples (panel a) and non-lung associatedsamples (panel b); non-lung associated samples include liver, kidney,and spleen.

FIG. 20 (panels a, b, c, d, e, and f) shows the bacterial burden in alllung-draining lymph nodes (panel a), as well as specific lung draininglymph nodes (panels b, c, and d); also included is the granuloma scorefor the carinal (panel e) and hilar (panel f) lymph nodes.

FIG. 21 shows the T cell response against each antigen is shown atvarious time points throughout the vaccination phase.

FIG. 22 shows a correlation between the CD4 T cell response against eachantigen, shown at various time points throughout the vaccination phase,and the extent of extrapulmonary spread.

FIG. 23 shows a correlation between the CD8 T cell response against eachantigen, shown at various time points throughout the vaccination phase,and the extent of extrapulmonary spread.

FIG. 24 (panels a, b, c, and d) shows infection by detection of de novoimmune responses. PBMCs were stimulated with RhCMV lysate (panels a andb) or CFP peptides (panels c and d); responses were analyzed byintracellular cytokine staining; shown are the percentages of memorycells expressing either IFNγ or TNF; included are responses fromperipheral blood mononuclear cells; CD4+(panels a and c) and CD8 (panelsb and d) T cells responses are shown.

FIG. 25 (panels a, b, c, d, e, and f) shows RhCMV-induced immuneresponses analyzed post-challenge; PBMCs were stimulated with ESAT6(panels a and b), Ag85B (panels c and d), and Rv1733 (panels e and f);responses were analyzed by intracellular cytokine staining; shown arethe percentages of memory cells expressing either IFNγ or TNF;CD4+(panels a, c, and e) and CD8+(panels b, d, and f) T cells responses.

FIG. 26 (panels a, b, c, and d) show the total RhCMV-induced immuneresponses in various compartments analyzed post-necropsy (panels a andb) and de novo CFP10 responses induced by infection (panels c and d);shown above are the percentages of memory cells expressing either IFNγor TNF.

FIG. 27 shows individual antigen-specific CD4+ T cell responses invarious compartments analyzed post-necropsy; shown above are thepercentages of memory cells expressing either IFNγ or TNF.

FIG. 28 shows individual antigen-specific CD4+ T cell responses invarious lymph nodes analyzed post-necropsy; shown above are thepercentages of memory cells expressing either IFNγ or TNF.

FIG. 29 shows the total RhCMV-induced immune responses in variouscompartments analyzed post-necropsy; panels c and d show de novo CFP10responses induced by infection; shown above are the percentages ofmemory cells expressing either IFNγ or TNF.

FIG. 30 shows the individual antigen-specific CD8+ T cell responses invarious compartments analyzed post-necropsy; shown above are thepercentages of memory cells expressing either IFNγ or TNF.

FIG. 31 shows individual antigen-specific CD8+ T cell responses invarious lymph nodes analyzed post-necropsy; shown above are thepercentages of memory cells expressing either IFNγ or TNF.

FIG. 32 shows a correlate analysis comparing the splenic CD4+ T cellresponse with the number of culture-positive lymph nodes.

FIG. 33 shows immune responses induced by vaccination analyzed byintracellular cytokine staining throughout the vaccination period; shownare the percentages of memory cells expressing either IFNγ or TNF;included are responses from peripheral blood mononuclear cells; CD4+ Tcells and CD8+ T cells are shown; the data points represent a summationof the antigens assayed, which are indicated below the panel.

FIG. 34 shows immune responses induced by vaccination normalized betweenvectors by summing only those antigens present in each vector (antigensnoted below the panel); responses were analyzed by intracellularcytokine staining throughout the vaccination period; shown are thepercentages of memory cells expressing either IFNγ or TNF; included areresponses from peripheral blood mononuclear cells; CD4+ T cells and CD8+T cells are shown.

FIG. 35 (panels a and b) shows immune responses induced by vaccinationanalyzed by intracellular cytokine staining throughout the vaccinationperiod; shown are the percentages of memory cells expressing either IFNγor TNF; included are responses from BAL; CD4+ T cells are shown in panela and CD8+ T cells are shown in panel b; the data points represent asummation of the antigens assayed, which are indicated below the panel.

FIG. 36 shows peripheral blood CFP10-specific T cell responses post-Mtbchallenge.

FIG. 37 shows clinical outcome data from longitudinal CT scans, which isused to quantify the volume of lung disease present post-challenge.

FIG. 38 shows a necropsy score.

FIG. 39 shows a culture score.

FIG. 40 shows overall efficacy of Strain 68-1 RhCMV/TB vectors.

FIG. 41 shows late disease resolution in a Strain 68-1 RhCMV/TB-6Agsingle vector vaccination.

FIG. 42 (panels a, b, c, d, e, and f) shows the immunogenicity ofRhCMV/TB and BCG vaccines for Study 3.

FIG. 43 (panels a, b, c, d, e, and f) shows the outcome of Mtb challengefor Study 3.

FIG. 44 (panels a, b, c, d, e, f, and g) shows the immunogenicity ofRhCMV/TB vaccines for Study 4.

FIG. 45 (panels a, b, c, d, e, f, g, and h) shows the outcome of Mtbchallenge for Study 4 and overall.

FIG. 46 (panels a, b, and c) shows RhCMV/TB vectors used in Study 3 and4.

FIG. 47 (panels a, b, and c) shows the development of de novoMtb-specific CD8+ T cell responses after Mtb challenge.

FIG. 48 shows pathologic scoring of TB disease at necropsy.

FIG. 49 (panels a, b, c, d, and e) shows a summary of outcomestatistics.

FIG. 50 (panels a, b, c, and d) shows a comparison of different measuresof TB infection outcome.

FIG. 51 shows a summary of TB disease outcome at necropsy of Study 3.

FIG. 52 shows MHC-restriction analysis of RhCMV/TB vector-elicited CD8+T cell responses in Study 4.

FIG. 53 (panels a and b) shows an analysis of non-vaccine-elicited,Mtb-specific CD4+ and CD8+ T cell responses at necropsy.

FIG. 54 shows a summary of outcome at necropsy of Study 4.

FIG. 55 (panels a, b, and c) shows immune correlates analysis of Studies3 and 4.

DESCRIPTION OF EMBODIMENTS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

As used herein, “adjuvant” means any molecule added to any compositiondescribed herein to enhance the immunogenicity of the Mtb antigens.

As used herein, “coding sequence” or “encoding nucleic acid” means thenucleic acids (RNA or DNA molecule) that comprise a nucleotide sequencewhich encodes an Mtb antigen. The coding sequence can further includeinitiation and termination signals operably linked to regulatoryelements including a promoter and polyadenylation signal capable ofdirecting expression in the cells of an individual or mammal to whichthe nucleic acid is administered.

As used herein, “consensus” or “consensus sequence” means a polypeptidesequence based on analysis of an alignment of multiple subtypes of aparticular Mtb antigen. Nucleic acid sequences that encode a consensuspolypeptide sequence can be prepared. Vaccines comprising Mtb antigensthat comprise consensus sequences and/or nucleic acid molecules thatencode such antigens can be used to induce broad immunity againstmultiple subtypes or serotypes of a particular antigen. I someembodiments, the consensus sequence may be the most common sequence.

As used herein, “electroporation” means the use of a transmembraneelectric field pulse to induce microscopic pathways (pores) in abio-membrane; their presence allows biomolecules such as plasmids,oligonucleotides, siRNA, drugs, ions, and water to pass from one side ofthe cellular membrane to the other.

As used herein, “fragment” with respect to nucleic acid sequences, meansa nucleic acid sequence or a portion thereof, that encodes a portion ofan Mtb antigen capable of eliciting an immune response in a mammal thatcross reacts with a full length wild type Mtb antigen. The fragments canbe DNA fragments selected from at least one of the various nucleotidesequences that encode protein fragments set forth below. For example,polynucleotides may comprise at least about 15, 20, 30, 40, 50, 75, 100,150, 200, 300, 400, 500 or 1000 or more contiguous nucleotides of one ormore of the sequences disclosed herein as well as all intermediatelengths there between. It will be readily understood that “intermediatelengths”, in this context, means any length between the quoted values,such as 16, 17, 18, 19, etc.; 21, 22, 23, etc.; 30, 31, 32, etc.; 50,51, 52, 53, etc.; 100, 101, 102, 103, etc.; 150, 151, 152, 153, etc.;including all integers through 200 to 500; 500 to 1,000, and the like.

As used herein, “fragment” or “immunogenic fragment” with respect topolypeptide sequences, means a portion of an MTB antigen capable ofeliciting an immune response in a mammal that cross reacts with a fulllength wild type strain Mtb antigen. Fragments of consensus or wild typeMtb antigens can comprise at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or at least 95% of a consensus or wild type Mtb antigen. Insome embodiments, fragments of consensus proteins can comprise at least20 amino acids or more, at least 30 amino acids or more, at least 40amino acids or more, at least 50 amino acids or more, at least 60 aminoacids or more, at least 70 amino acids or more, at least 80 amino acidsor more, at least 90 amino acids or more, at least 100 amino acids ormore, at least 110 amino acids or more, at least 120 amino acids ormore, at least 130 amino acids or more, at least 140 amino acids ormore, at least 150 amino acids or more, at least 160 amino acids ormore, at least 170 amino acids or more, at least 180 amino acids or moreof a consensus or wild type protein.

As used herein, “genetic construct” refers to the DNA or RNA moleculesthat comprise a nucleotide sequence which encodes an Mtb antigen. Thecoding sequence includes initiation and termination signals operablylinked to regulatory elements including a promoter and polyadenylationsignal capable of directing expression in the cells of the individual towhom the nucleic acid molecule is administered.

As used herein, “expressible form” refers to gene constructs thatcontain the necessary regulatory elements operable linked to a codingsequence that encodes an Mtb antigen such that when present in the cellof the individual, the coding sequence will be expressed.

As used herein, “homology” refers to a degree of complementarity fornucleic acid molecules. There can be partial homology or completehomology (i.e., identity). A partially complementary sequence that atleast partially inhibits a completely complementary sequence fromhybridizing to a target nucleic acid is referred to using the functionalterm “substantially homologous.” When used in reference to adouble-stranded nucleic acid sequence such as a cDNA or genomic clone,the term “substantially homologous” refers to a probe that can hybridizeto a strand of the double-stranded nucleic acid sequence underconditions of low stringency. When used in reference to asingle-stranded nucleic acid sequence, the term “substantiallyhomologous” refers to a probe that can hybridize to (i.e., is thecomplement of) the single-stranded nucleic acid template sequence underconditions of low stringency.

As used herein, “identical” or “identity” in the context of two or morenucleic acids or polypeptide sequences, means that the sequences have aspecified percentage of residues that are the same over a specifiedregion. The percentage can be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) residues can be consideredequivalent. Identity and/or homology can be performed manually or byusing a computer sequence algorithm such as BLAST or BLAST 2.0.

As used herein, “immune response” means the activation of a host'simmune system, e.g., that of a mammal, in response to the introductionof an Mtb antigen. The immune response can be in the form of a cellularor humoral response, or both.

As used herein, “isolated” means that a polynucleotide is substantiallyaway from other coding sequences, and that the nucleic acid segment doesnot contain large portions of unrelated coding DNA, such as largechromosomal fragments or other functional genes or polypeptide codingregions. Of course, this refers to the nucleic acid segment asoriginally isolated, and does not exclude genes or coding regions lateradded to the segment by the hand of man.

As used herein, “Mtb antigen” means an antigen from Mycobacteriumtuberculosis, which may be an isolated antigen, or an antigen that formspart of a fusion protein with other antigen(s).

As used herein, “Mycobacteria” means a genus of aerobic intracellularbacterial organisms. Upon invasion of a host, these organisms survivewithin endosomal compartments of monocytes and macrophages. Humanmycobacterial diseases include tuberculosis (caused by M. tuberculosis(Mtb)), Leprosy (caused by M. leprae), Bairnsdale ulcers (caused by M.ulcerans), and other infections that can be caused by M. marinum, M.kansasii, M. scrofulaceum, M szulgai, M xenopi, M. fortuitum, Mhaemophilum, M chelonei, and M. intracelluare. Mycobacterium strainsthat were previously considered to be nonpathogenic (such as M. avium)are also now known to be major killers of immunosuppressed AIDSpatients. The major response to mycobacteria involves cell mediatedhypersensitivity (DTH) reactions with T cells and macrophages playingmajor roles in the intracellular killing and walling off (or containing)of the organism (granuloma formation). A major T cell response involvesCD4⁺ lymphocytes that recognize mycobacterial heat shock proteins andimmunodominant antigens.

As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide”means at least two nucleotides covalently linked together, which hasbeen isolated free of total genomic DNA of a particular species.Included within these terms are nucleic acid segments and smallerfragments of such segments, and also recombinant CMV vectors. Thedepiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid can be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that canhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions. Nucleic acids can be single strandedor double stranded, or can contain portions of both double stranded andsingle stranded sequence. The nucleic acid can be DNA, both genomic andcDNA, RNA, or a hybrid, where the nucleic acid can contain combinationsof deoxyribo- and ribo-nucleotides, and combinations of bases includinguracil, adenine, thymine, cytosine, guanine, inosine, xanthinehypoxanthine, isocytosine and isoguanine. Nucleic acids can be obtainedby chemical synthesis methods or by recombinant methods. As will beunderstood by those skilled in the art, the nucleic acid molecules caninclude genomic sequences, extra-genomic and plasmid-encoded sequencesand smaller engineered gene segments that express, or may be adapted toexpress, proteins, polypeptides, peptides and the like. Such segmentsmay be naturally isolated, or modified synthetically by the hand of man.

As used herein, “operably linked” means that expression of a gene isunder the control of a promoter with which it is spatially connected. Apromoter can be positioned 5′ (upstream) or 3′ (downstream) of a geneunder its control. The distance between the promoter and a gene can beapproximately the same as the distance between that promoter and thegene it controls in the gene from which the promoter is derived. As isknown in the art, variation in this distance can be accommodated withoutloss of promoter function.

As used herein, “promoter” means a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter can comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter can also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter can bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter can regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents.

As used herein, “signal peptide” and “leader sequence”, usedinterchangeably, refer to an amino acid sequence that can be linked atthe amino terminus of an Mtb antigenic protein set forth herein. Signalpeptides/leader sequences typically direct localization of a protein.Signal peptides/leader sequences used herein can facilitate secretion ofthe protein from the cell in which it is produced or anchor it in themembrane. Signal peptides/leader sequences are often cleaved from theremainder of the protein, often referred to as the mature protein, uponsecretion from the cell. Signal peptides/leader sequences are linked atthe N terminus of the protein.

As used herein, “stringent hybridization conditions” means conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionscan be selected to be about 5 to 10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) can be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions can be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10 to 50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions can alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal can be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

As used herein, “substantially complementary” means that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the complement of a second sequence over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360,450, 540, or more nucleotides or amino acids, or that the two sequenceshybridize under stringent hybridization conditions.

As used herein, “substantially identical” means that a first and secondsequence are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 180, 270, 360, 450, 540 or morenucleotides or amino acids, or with respect to nucleic acids, if thefirst sequence is substantially complementary to the complement of thesecond sequence.

As used herein, “tuberculosis” means a disease that is generally causedby Mycobacterium tuberculosis that usually infects the lungs. However,other “atypical” mycobacteria such as M. kansasii may produce a similarclinical and pathologic appearance of disease. Transmission of M.tuberculosis occurs by the airborne route in confined areas with poorventilation. In more than 90% of cases, following infection with M.tuberculosis, the immune system prevents development of disease from M.tuberculosis, often called, active tuberculosis. However, not all of theM. tuberculosis is killed and, thus tiny, hard capsules are formed.“Primary tuberculosis” is seen as disease that develops following aninitial infection, usually in children. The initial focus of infectionis a small subpleural granuloma accompanied by granulomatous hilar lymphnode infection. Together, these make up the Ghon complex. In nearly allcases, these granulomas resolve and there is no further spread of theinfection. “Secondary tuberculosis” is seen mostly in adults as areactivation of previous infection (or reinfection), particularly whenhealth status declines. The granulomatous inflammation is much moreflorid and widespread. Typically, the upper lung lobes are mostaffected, and cavitation can occur. Dissemination of tuberculosisoutside of the lungs can lead to the appearance of a number of uncommonfindings with characteristic patterns that include skeletaltuberculosis, genital tract tuberculosis, urinary tract tuberculosis,central nervous system (CNS) tuberculosis, gastrointestinaltuberculosis, adrenal tuberculosis, scrofula, and cardiac tuberculosis.“Latent” tuberculosis is an Mtb infection in an individual that can bedetected by a diagnostic assay, such as, but not limited to a tuberculinskin test (TST) wherein the infection does not produce symptoms in thatindividual. “Active” tuberculosis is a symptomatic Mtb infection in asubject. Microscopically, the inflammation produced with TB infection isgranulomatous, with epithelioid macrophages and Langhans giant cellsalong with lymphocytes, plasma cells, maybe a few polymorphonuclearcells, fibroblasts with collagen, and characteristic caseous necrosis inthe center. The inflammatory response is mediated by a type IVhypersensitivity reaction, and skin testing is based on this reaction.In some examples, tuberculosis can be diagnosed by a skin test, an acidfast stain, an auramine stain, or a combination thereof. The most commonspecimen screened is sputum, but the histologic stains can also beperformed on tissues or other body fluids.

As used herein, “variant” with respect to a nucleic acid means: i) aportion or fragment of a referenced nucleotide sequence; ii) thecomplement of a referenced nucleotide sequence or portion thereof; iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

As used herein, “variant” with respect to a peptide or polypeptide meansthat it differs in amino acid sequence by the insertion, deletion, orconservative substitution of amino acids, but retains at least onebiological activity. Variant can also mean a protein with an amino acidsequence that is substantially identical to a referenced protein with anamino acid sequence that retains at least one biological activity. Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity, degree and distribution of charged regions) isrecognized in the art as typically involving a minor change Amino acidsubstitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties. Theterm “variant” also encompasses homologous genes of xenogeneic origin.

As used herein, “CMV vector” means a CMV nucleic acid molecule asintroduced into a host cell, thereby producing a transformed host cell.A CMV vector may include nucleic acid sequences that permit it toreplicate in a host cell, such as an origin of replication. A CMV vectormay also include one or more selectable marker gene and other geneticelements known in the art.

The present disclosure provides recombinant RhCMV or HCMV vectorscomprising a nucleic acid molecule encoding an expressible Mtb antigenselected from Ag85A-Ag85B-Rv3407, Rv1733-Rv2626c, RpfA-RpfC-RpfD,Ag85B-ESAT6, and Ag85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD. In someembodiments, the nucleic acid molecule encoding any particular Mtbantigen can be a mycobacterial sequence, a bacterial codon optimizedsequence (such as an E. coli optimized sequence), or a mammalianoptimized sequence (such as a human optimized sequence). Methods ofcodon optimization (whether for bacterial or mammalian) are well knownto the skilled artisan.

In any of the embodiments of the nucleic acid molecules set forthherein, the individual Mtb nucleic acid sequences can be present in anyorder. For example, for a fusion protein comprising Ag85A, Ag85B, andRv3407 antigens, the first (or N-terminal) nucleic acid molecule mayencode Ag85A, Ag85B, or Rv3407; the second nucleic acid molecule mayencode Ag85A, Ag85B, or Rv3407 (whichever one is not the first Mtbantigen); and the third nucleic acid molecule may encode Ag85A, Ag85B,or Rv3407 (whichever one is not the first or second Mtb antigen).Likewise for every nucleic acid molecule disclosed herein.

Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide of the present invention, and a polynucleotidemay, but need not, be linked to other molecules and/or supportmaterials.

Polynucleotides may comprise a native sequence (e.g., an endogenoussequence that encodes a CMV or TB protein or a portion thereof) or maycomprise a variant, or a biological or antigenic functional equivalentof such a sequence.

A nucleotide sequence encoding Ag85A is shown in Table 1 as SEQ ID NO:1,and an amino acid sequence of Ag85A is shown in Table 1 as SEQ ID NO:2.

A nucleotide sequence encoding Ag85B is shown in Table 1 as SEQ ID NO:3,and an amino acid sequence of Ag85B is shown in Table 1 as SEQ ID NO:4.

A nucleotide sequence encoding Rv3407 is shown in Table 1 as SEQ IDNO:5, and an amino acid sequence of Rv3407 is shown in Table 1 as SEQ IDNO:6.

A nucleotide sequence encoding Rv1733 is shown in Table 1 as SEQ IDNO:7, and an amino acid sequence of Rv1733 is shown in Table 1 as SEQ IDNO:8.

A nucleotide sequence encoding Rv2626c is shown in Table 1 as SEQ IDNO:9, and an amino acid sequence of Rv2626c is shown in Table 1 as SEQID NO:10.

A nucleotide sequence encoding RpfA is shown in Table 1 as SEQ ID NO:11,and an amino acid sequence of RpfA is shown in Table 1 as SEQ ID NO:12.

A nucleotide sequence encoding RpfC is shown in Table 1 as SEQ ID NO:13,and an amino acid sequence of RpfC is shown in Table 1 as SEQ ID NO:14.

A nucleotide sequence encoding RpfD is shown in Table 1 as SEQ ID NO:15,and an amino acid sequence of RpfD is shown in Table 1 as SEQ ID NO:16.

A nucleotide sequence encoding ESAT-6 is shown in Table 1 as SEQ IDNO:17, and an amino acid sequence of ESAT-6 is shown in Table 1 as SEQID NO:18.

TABLE 1 nucleotide sequence Construct amino acid sequence Ag85Aatgcagcttgagacagggttcgtggcgccgtcacgggtatgtcgcgtcgactcgtggtcggggccgtcggcgcggccctagtgtcgggtctggtcggcgccgtcggtggcacggcgaccgcgggggcattacccggccgggcttgccggtggagtacctgcaggtgccgtcgccgtcgatgggccgtgacatcaaggtccaattccaaagtggtggtgccaactcgcccgccctgtacctgctcgacggcctgcgcgcgcaggacgacttcagcggctgggacatcaacaccccggcgttcgagtggtacgaccagtcgggcctgtcggtggtcatgccggtgggtggccagtcaagatctactccgactggtaccagcccgcctgcggcaaggccggagccagacttacaagtgggagaccacctgaccagcgagctgccggggtggctgcaggccaacaggcacgtcaagcccaccggaagcgccgtcgtcggtattcgatggctgatatcggcgctgacgctggcgatctatcacccccagcagttcgtctacgcgggagcgatgtcgggcctgaggacccctcccaggcgatgggtcccaccctgatcggcctggcgatgggtgacgctggcggctacaaggcctccgacatgtggggcccgaaggaggacccggcgtggcagcgcaacgacccgctgttgaacgtcgggaagctgatcgccaacaacacccgcgtctgggtgtactgcggcaacggcaagccgtcggatctgggtggcaacaacctgccggccaagacctcgagggcttcgtgcggaccagcaacatcaagaccaagacgcctacaacgccggtggcggccacaacggcgtgacgacttcccggacagcggtacgcacagctgggagtactggggcgcgcagctcaacgctatgaagcccgacctgcaacgggcactgggtgccacgcccaacaccgggcccgcgccccagggcgcctag (SEQ ID NO: 1)MQLVDRVRGAVTGMSRRLVVGAVGAALVSGLVGAVGGTATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGA (SEQ ID NO: 2) Ag85Batgacagacgtgagccgaaagattcgagcaggggacgccgattgatgatcggcacggcagcggctgtagtccaccgggcctggtggggcttgccggcggagcggcaaccgcgggcgcgttctcccggccggggctgccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgcgacatcaaggttcagttccagagcggtgggaacaactcacctgcggtttatctgctcgacggcctgcgcgcccaagacgactacaacggctgggatatcaacaccccggcgttcgagtggtactaccagtcgggactgtcgatagtcatgccggtcggcgggcagtccagatctacagcgactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaaaccacctgaccagcgagctgccgcaatggagtccgccaacagggccgtgaagcccaccggcagcgctgcaatcggcttgtcgatggccggctcgtcggcaatgatcttggccgcctaccacccccagcagttcatctacgccggctcgctgtcggccctgctggacccctctcaggggatggggcctagcctgatcggcctcgcgatgggtgacgccggcggttacaaggccgcagacatgtggggtccctcgagtgacccggcatgggagcgcaacgaccctacgcagcagatccccaagctggtcgcaaacaacacccggctatgggatattgcgggaacggcaccccgaacgagagggcggtgccaacatacccgccgagttcaggagaacttcgttcgtagcagcaacctgaagttccaggatgcgtacaacgccgcgggcgggcacaacgccgtgacaacttcccgcccaacggcacgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagttcgttaggcgccggctga (SEQ ID NO: 3)MTDVSRKIRAWGRRLMIGTAAAVVLPGLVGLAGGAATAGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAG (SEQ ID NO: 4) Rv3407atgcgtgctaccgagggcagtggaggcaatcggaatccgagaactaagacagcacgcatcgcgatacctcgcccgggagaagccggcgaggaacttggcgtcaccaacaaaggaagacttgtggcccgactcatcccggtgcaggccgcggagcgactcgcgaagccctgattgaatcaggtgtcctgattccggctcgtcgtccacaaaaccttctcgacgtcaccgccgaaccggcgcgcggccgcaagcgcaccctgtccgatgttctcaacgaaatgcgcgacgagcagtga (SEQ ID NO: 5)MRATVGLVEAIGIRELRQHASRYLARVEAGEELGVTNKGRLVARLIPVQAAERSREALIESGVLIPARRPQNLLDVTAEPARGRKRTLSDVLNEMRDEQ (SEQ ID NO: 6) Rv1733atgatcgccacaacccgcgatcgtgaaggagccaccatgatcacgataggctgcgcttgccgtgccggacgatactgcgggtgacagccgcaatccgctggtgcgtgggacggatcgactcgaggcggtcgtcatgctgctggccgtcacggtctcgctgctgactatcccgttcgccgccgcggccggcaccgcagtccaggattcccgcagccacgtctatgcccaccaggcccagacccgccatcccgcaaccgcgaccgtgatcgatcacgagggggtgatcgacagcaacacgaccgccacgtcagcgccgccgcgcacgaagatcaccgtgcctgcccgatgggtcgtgaacggaatagaacgcagcggtgaggtcaacgcgaagccgggaaccaaatccggtgaccgcgtcggcatttgggtcgacagtgccggtcagctggtcgatgaaccagctccgccggcccgtgccattgcggatgcggccctggccgccttgggactctggttgagcgtcgccgcggttgcgggcgccctgctggcgctcactcgggcgattctgatccgcgttcgcaacgccagaggcaacacgacatcgacagcctgactgcacgcagcggtga (SEQ ID NO: 7)MIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQR (SEQ ID NO: 8) Rv2626catgaccaccgcacgcgacatcatgaacgcaggtgtgacctgtgttggcgaacacgagacgctaaccgctgccgctcaatacatgcgtgagcacgacatcggcgcgttgccgatctgcggggacgacgaccggctgcacggcatgctcaccgaccgcgacattgtgatcaaaggcctggctgcgggcctagacccgaataccgccacggctggcgagaggcccgggacagcatctactacgtcgatgcgaacgcaagcatccaggagatgctcaacgtcatggaagaacatcaggtccgccgtgaccggtcatctcagagcaccgcaggtcggaatcgtcaccgaagccgacatcgcccgacacctgcccgagcacgccattgtgcagttcgtcaaggcaatctgctcgcccatggccctcgccagctag (SEQ ID NO: 9)MTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALAS (SEQ ID NO: 10) RpfAatgagtggacgccaccgtaagcccaccacatccaacgtcagcgtcgccaagatcgcattaccggcgcagtactcggtggcggcggcatcgccatggccgctcaggcgaccgcggccaccgacggggaatgggatcaggtggcccgctgcgagtcgggcggcaactggtcgatcaacaccggcaacggttacctcggtggcttgcagttcactcaaagcacctgggccgcacatggtggcggcgagttcgccccgtcggctcagctggccagccgggagcagcagattgccgtcggtgagcgggtgctggccacccagggtcgcggcgcctggccggtgtgcggccgcgggttatcgaacgcaacaccccgcgaagtgatcccgcttcggcagcgatggacgctccgaggacgcggccgcggtcaacggcgaaccagcaccgctggccccgccgcccgccgacccggcgccacccgtggaacttgccgctaacgacctgcccgcaccgctgggtgaacccctcccggcagctcccgccgacccggcaccacccgccgacctggcaccacccgcgcccgccgacgtcgcgccacccgtggaacttgccgtaaacgacctgcccgcaccgctgggtgaacccctcccggcagctcccgccgacccggcaccacccgccgacctggcaccacccgcgcccgccgacctggcgccacccgcgcccgccgacctggcgccacccgcgcccgccgacctggcaccacccgtggaacttgccgtaaacgacctgcccgcgccgctgggtgaacccctcccggcagctcccgccgaactggcgccacccgccgatctggcacccgcgtccgccgacctggcgccacccgcgcccgccgacctggcgccacccgcgcccgccgaactggcgccacccgcgcccgccgacctggcaccacccgctgcggtgaacgagcaaaccgcgccgggcgatcagcccgccacagctccaggcggcccggaggccagccaccgataggaactccccgagcccgacccccaaccagctgacgcaccgccgcccggcgacgtcaccgaggcgcccgccgaaacgccccaagtctcgaacatcgcctatacgaagaagctgtggcaggcgattcgggcccaggacgtctgcggcaacgatgcgctggactcgctcgcacagccgtacgtcatcggctga (SEQ ID NO: 11)MSGRHRKPTTSNVSVAKIAFTGAVLGGGGIAMAAQATAATDGEWDQVARCESGGNWSINTGNGYLGGLQFTQSTWAAHGGGEFAPSAQLASREQQIAVGERVLATQGRGAWPVCGRGLSNATPREVLPASAAMDAPLDAAAVNGEPAPLAPPPADPAPPVELAANDLPAPLGEPLPAAPADPAPPADLAPPAPADVAPPVELAVNDLPAPLGEPLPAAPADPAPPADLAPPAPADLAPPAPADLAPPAPADLAPPVELAVNDLPAPLGEPLPAAPAELAPPADLAPASADLAPPAPADLAPPAPAELAPPAPADLAPPAAVNEQTAPGDQPATAPGGPVGLATDLELPEPDPQPADAPPPGDVTEAPAETPQVSNIAYTKKLWQAIRAQDVCGNDALDSLAQPYVIG (SEQ ID NO: 12) RpfCgtgcatcattgccggccgaccacggccggtcgcggtgcaatagacacccgatctcaccactctctctaatcggtaacgcttcggccacttccggcgatatgtcgagcatgacaagaatcgccaagccgctcatcaagtccgccatggccgcaggactcgtcacggcatccatgtcgctctccaccgccgttgcccacgccggtcccagcccgaactgggacgccgtcgcgcagtgcgaatccgggggcaactgggcggccaacaccggaaacggcaaatacggcggactgcagttcaagccggccacctgggccgcattcggcggtgtcggcaacccagcagctgcctctcgggaacaacaaatcgcagagccaatcgggactcgccgaacagggattggacgcgtggccgacgtgcggcgccgcctctggccaccgatcgcactgtggtcgaaacccgcgcagggcatcaagcaaatcatcaacgagatcatttgggcaggcattcaggcaagtattccgcgctga (SEQ ID NO: 13)VHPLPADHGRSRCNRHPISPLSLIGNASATSGDMSSMTRIAKPLIKSAMAAGLVTASMSLSTAVAHAGPSPNWDAVAQCESGGNWAANTGNGKYGGLQFKPATWAAFGGVGNPAAASREQQIAVANRVLAEQGLDAWPTCGAASGLPIALWSKPAQGIKQIINEIIWAGIQASIPR (SEQ ID NO: 14)RpfD atgacaccgggatgcttactactgcgggtgctggccgaccacgtgacaggtgcgccaggatcgtatgcacggtgacatcgaaaccgccgagtcgcgaccatgatgtcgcgttgagggtctgtccaccatcagctcgaaagccgacgacatcgattgggacgccatcgcgcaatgcgaatccggcggcaattgggcggccaacaccggtaacgggttatacggtggtctgcagatcagccaggcgacgtgggattccaacggtggtgtcgggtcgccggcggccgcgagtccccagcaacagatcgaggtcgcagacaacattatgaaaacccaaggcccgggtgcgtggccgaaatgtagacttgtagtcagggagacgcaccgctgggctcgctcacccacatcctgacgacctcgcggccgagactggaggttgttcggggagcagggacgattga (SEQ ID NO: 15)MTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 16) ESAT-6atgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgacgcatag (SEQ ID NO: 17)MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO: 18)

All sequences shown in Table 1 are derived from HCMV.

In some embodiments, the fusion protein comprises Ag85A, Ag85B, andRv3407 antigens. In some embodiments, the fusion protein comprisesRv1733 and Rv2626c antigens. In some embodiments, the fusion proteincomprises RpfA, RpfC, and Rpm antigens. In some embodiments, the fusionprotein comprises Ag85B and ESAT6 antigens. In some embodiments, thefusion protein comprises Ag85A, ESAT6, Rv3407, Rv2626c, RpfA, and Rpmantigens.

In any of the embodiments of fusion proteins set forth herein, theindividual Mtb antigens can be present in any order. For example, for afusion protein comprising Ag85A, Ag85B, and Rv3407 antigens, the first(or N-terminal) antigen may be Ag85A, Ag85B, or Rv3407; the secondantigen may be Ag85A, Ag85B, or Rv3407 (whichever one is not the firstMtb antigen); and the third antigen may be Ag85A, Ag85B, or Rv3407(whichever one is not the first or second Mtb antigen). Likewise forevery fusion protein disclosed herein.

Individual Mtb antigens may be linked together in a C-terminus toN-terminus or N-terminus to C-terminus manner without any linker.Alternately, a linker may be present between any two Mtb antigens withinany of the fusion proteins disclosed herein. In some embodiments, thelinker is a segment of DNA optionally containing one or morerestrictions sites, wherein the linker is inserted between nucleic acidmolecules encoding two Mtb antigens of any of the fusion proteinsdisclosed herein.

In some embodiments, the fusion protein comprises Ag85A-Ag85B-Rv3407(Construct A; see Table 2). The nucleotide sequence is SEQ ID NO:19, andthe corresponding amino acid sequence is SEQ ID NO:20.

In some embodiments, the fusion protein comprises Rv1733-Rv2626c(Construct B; see Table 2). The nucleotide sequence is SEQ ID NO:21, andthe corresponding amino acid sequence is SEQ ID NO:22.

In some embodiments, the fusion protein comprises RpfA-RpfC-RpfD(Construct C; see Table 2). The nucleotide sequence is SEQ ID NO:23, andthe corresponding amino acid sequence is SEQ ID NO:24.

In some embodiments, the fusion protein comprises Ag85B-ESAT6 (ConstructD; see Table 2). The nucleotide sequence is SEQ ID NO:25, and thecorresponding amino acid sequence is SEQ ID NO:26.

In some embodiments, the fusion protein comprisesAg85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD (Construct E; see Table 2). Thenucleotide sequence is SEQ ID NO:27 or SEQ ID NO:28, and thecorresponding amino acid sequence is SEQ ID NO:29 or SEQ ID NO:30.

TABLE 2 Construct nucleotide sequence amino acid sequence Aatggcattacccggccgggcttgccggtggagtacctgcaggtgccgtcgccgtcgatgggccgtgacatcaaggtccaattccaaagtggtggtgccaactcgcccgccctgtacctgctcgacggcctgcgcgcgcaggacgacttcagcggctgggacatcaacaccccggcgttcgagtggtacgaccagtcgggcctgtcggtggtcatgccggtgggtggccagtcaagcttctactccgactggtaccagcccgcctgcggcaaggccggttgccagacttacaagtgggagaccttcctgaccagcgagctgccggggtggctgcaggccaacaggcacgtcaagcccaccggaagcgccgtcgtcggtattcgatggctgatatcggcgctgacgctggcgatctatcacccccagcagttcgtctacgcgggagcgatgtcgggcctgttggacccctcccaggcgatgggtcccaccctgatcggcctggcgatgggtgacgctggcggctacaaggcctccgacatgtggggcccgaaggaggacccggcgtggcagcgcaacgacccgctgttgaacgtcgggaagctgatcgccaacaacacccgcgtctgggtgtactgcggcaacggcaagccgtcggatctgggtggcaacaacctgccggccaagacctcgagggcttcgtgcggaccagcaacatcaagttccaagacgcctacaacgccggtggcggccacaacggcgtgacgacttcccggacagcggtacgcacagctgggagtactggggcgcgcagctcaacgctatgaagcccgacctgcaacgggcactgggtgccacgcccaacaccgggcccgcgccccagggcgccatgactcccggccggggctgccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgcgacatcaaggttcagttccagagcggtgggaacaactcacctgcggatatctgctcgacggcctgcgcgcccaagacgactacaacggctgggatatcaacaccccggcgttcgagtggtactaccagtcgggactgtcgatagtcatgccggtcggcgggcagtccagatctacagcgactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaaaccacctgaccagcgagctgccgcaatggagtccgccaacagggccgtgaagcccaccggcagcgctgcaatcggcttgtcgatggccggctcgtcggcaatgatcaggccgcctaccacccccagcagttcatctacgccggctcgctgtcggccctgctggacccctctcaggggatggggcctagcctgatcggcctcgcgatgggtgacgccggcggttacaaggccgcagacatgtggggtccctcgagtgacccggcatgggagcgcaacgaccctacgcagcagatccccaagctggtcgcaaacaacacccggctatgggatattgcgggaacggcaccccgaacgagagggcggtgccaacatacccgccgagttcaggagaacttcgttcgtagcagcaacctgaagaccaggatgcgtacaacgccgcgggcgggcacaacgccgtgacaacttcccgcccaacggcacgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagttcgttaggcgccggcatgcgtgctaccgagggcttgtggaggcaatcggaatccgagaactaagacagcacgcatcgcgatacctcgcccgggagaagccggcgaggaacttggcgtcaccaacaaaggaagacttgtggcccgactcatcccggtgcaggccgcggagcgttctcgcgaagccctgattgaatcaggtgtcctgattccggctcgtcgtccacaaaaccactcgacgtcaccgccgaaccggcgcgcggccgcaagcgcaccctgtccgatgttctcaacgaaatgcgcgacgagcagtga (SEQ ID NO: 19)MAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGAAARATVGLVEAIGIRELRQHASRYLARVEAGEELGVTNKGRLVARLIPVQAAERSREALIESGVLIPARRPQNLLDVTAEPARGRKRTLSDVLNEMRDEQ (SEQ ID NO: 20) Batgaccaccgcacgcgacatcatgaacgcaggtgtgacctgtgaggcgaacacgagacgctaaccgctgccgctcaatacatgcgtgagcacgacatcggcgcgttgccgatctgcggggacgacgaccggctgcacggcatgctcaccgaccgcgacattgtgatcaaaggcctggctgcgggcctagacccgaataccgccacggctggcgagaggcccgggacagcatctactacgtcgatgcgaacgcaagcatccaggagatgctcaacgtcatggaagaacatcaggtccgccgtgaccggtcatctcagagcaccgcaggtcggaatcgtcaccgaagccgacatcgcccgacacctgcccgagcacgccattgtgcagttcgtcaaggcaatctgctcgcccatggccctcgccagcatgatcgccacaacccgcgatcgtgaaggagccaccatgatcacgataggctgcgcttgccgtgccggacgatactgcgggtgacagccgcaatccgctggtgcgtgggacggatcgactcgaggcggtcgtcatgctgctggccgtcacggtctcgctgctgactatcccgttcgccgccgcggccggcaccgcagtccaggattcccgcagccacgtctatgcccaccaggcccagacccgccatcccgcaaccgcgaccgtgatcgatcacgagggggtgatcgacagcaacacgaccgccacgtcagcgccgccgcgcacgaagatcaccgtgcctgcccgatgggtcgtgaacggaatagaacgcagcggtgaggtcaacgcgaagccgggaaccaaatccggtgaccgcgtcggcatttgggtcgacagtgccggtcagctggtcgatgaaccagctccgccggcccgtgccattgcggatgcggccctggccgccttgggactctggttgagcgtcgccgcggttgcgggcgccctgctggcgctcactcgggcgattctgatccgcgttcgcaacgccagaggcaacacgacatcgacagcctgactgcacgcagcggtga (SEQ ID NO: 21)MTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASMIATTRDREGATMITFRLRLPCRTILRVFSRNPLVRGTDRLEAVVMLLAVTVSLLTIPFAAAAGTAVQDSRSHVYAHQAQTRHPATATVIDHEGVIDSNTTATSAPPRTKITVPARWVVNGIERSGEVNAKPGTKSGDRVGIWVDSAGQLVDEPAPPARAIADAALAALGLWLSVAAVAGALLALTRAILIRVRNASWQHDIDSLFCTQR(SEQ ID NO: 22) Catggcgtcagggaggcatcggaaaccaactacaagcaatgtatctgagccaagattgattcaccggcgcagttcttggaggtggcggaattgccatggctgcccaggcaacagccgctacagatggagagtgggatcaggtggctcgatgtgagtctggtggcaactggtctatcaacactgggaacgggtatcaggcggcttgcaatttactcagagcacttgggctgcccacggagggggtgaatttgctcctagcgcgcagctggcctcccgcgagcagcagatcgctgtgggagagagggtgaggccacacagggaagaggtgcctggcctgtctgtggccgcggactcagtaatgctacccctagggaggtgctgcccgcctcagccgctatggacgctccactggatgctgccgccgtgaatggcgagccagctccgctggcacccccacctgcagaccccgctcccccagtcgagctggcggcaaacgacctgcccgcacctctcggagaaccacttcctgcagcgcctgccgatccagctccacctgctgataggctccccccgctcccgccgatgtagcccctccggtcgagaggctgtgaatgacctgccggcacctctgggcgagcccctcccagccgctccggccgaccctgcccctcctgctgatctggcaccacccgctcctgccgacctcgccccacccgccccagcagacctggctccaccagcgcctgcggatcttgccccgcctgagagctggctgtcaacgatcttcctgcgcctcaggagagcccctgcccgctgctccagccgaactcgcaccaccggcagatctggctcccgcctctgccgatcttgcacctcccgcaccggcggacttggcacctccagcaccagcagaactggctccccctgcgccggctgacctggcccctccagcagccgttaatgagcaaaccgcaccaggggaccagccggctacggcaccaggtggaccggtggggctggccaccgacctggagctgcctgagccggatccccaaccagctgatgctcccccacctggcgacgtaactgaggccccagctgaaacgccccaggtcagtaacatcgcttacacaaagaaactgtggcaggcaattagggctcaggacgtgtgtgggaacgacgccctggacagcaggcccaaccgtacgtgatcggtatgcaccccctccccgctgatcatggtcgcagtcgctgtaaccgccaccccatttcacctctcagccttaagggaatgcgtctgctacaagtggcgacatgtctagtatgacaaggattgctaagcccctcatcaaaagtgcgatggctgccggtctggtaacagcatccatgagcttgtccaccgcagtggctcacgctgggccaccccgaactgggatgccgtcgcccagtgcgagtcaggcggcaattgggccgcaaataccggtaacggtaagtatggaggactgcagataaacctgcaacttgggccgcctaggaggagtgggtaatcctgcagctgatctagagaacagcagattgccgtggctaaccgcgttctcgcggagcagggtctggacgcctggccgacctgtggcgccgcatcaggatgccgatcgcgagtggtcaaagcccgcccagggaatcaagcagattatcaatgagatcatctgggccggaatacaggcaagcatccctagaatgactcctgggcactgacaaccgctggcgctgggaggcccagggataggtgcgcccggatcgtagtaccgtattcatagagaccgccgtggtcgcgacaatgacgtggctctcagggcagagcaccattagctctaaggccgatgatatagattgggatgctattgctcaatgcgaatccggtgggaactgggccgctaataccggaaatgggctctacggcggactgcagatcagccaggctacatgggatagcaacggaggagtcgggtcccctgccgctgcatccccgcaacagcaaatcgaggtggccgataacatcatgaaaacccagggacccggagcctggcccaaatgtagctcatgtagccaaggagatgcgcccctcggacactgacgcacatcctcaccacctcgccgcggaaaccggagggtgctctggcagccgggacgactga (SEQ ID NO: 23)MSGRHRKPTTSNVSVAKIAFTGAVLGGGGIAMAAQATAATDGEWDQVARCESGGNWSINTGNGYLGGLQFTQSTWAAHGGGEFAPSAQLASREQQIAVGERVLATQGRGAWPVCGRGLSNATPREVLPASAAMDAPLDAAAVNGEPAPLAPPPADPAPPVELAANDLPAPLGEPLPAAPADPAPPADLAPPAPADVAPPVELAVNDLPAPLGEPLPAAPADPAPPADLAPPAPADLAPPAPADLAPPAPADLAPPVELAVNDLPAPLGEPLPAAPAELAPPADLAPASADLAPPAPADLAPPAPAELAPPAPADLAPPAAVNEQTAPGDQPATAPGGPVGLATDLELPEPDPQPADAPPPGDVTEAPAETPQVSNIAYTKKLWQAIRAQDVCGNDALDSLAQPYVIGVHPLPADHGRSRCNRHPISPLSLIGNASATSGDMSSMTRIAKPLIKSAMAAGLVTASMSLSTAVAHAGPSPNWDAVAQCESGGNWAANTGNGKYGGLQFKPATWAAFGGVGNPAAASREQQIAVANRVLAEQGLDAWPTCGAASGLPIALWSKPAQGIKQIINEIIWAGIQASIPRMTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 24) Datgttctcccggccggggctgccggtcgagtacctgcaggtgccgtcgccgtcgatgggccgcgacatcaaggttcagttccagagcggtgggaacaactcacctgcggtttatctgctcgacggcctgcgcgcccaagacgactacaacggctgggatatcaacaccccggcgttcgagtggtactaccagtcgggactgtcgatagtcatgccggtcggcgggcagtccagcttctacagcgactggtacagcccggcctgcggtaaggctggctgccagacttacaagtgggaaaccttcctgaccagcgagctgccgcaatggttgtccgccaacagggccgtgaagcccaccggcagcgctgcaatcggcttgtcgatggccggctcgtcggcaatgatcttggccgcctaccacccccagcagttcatctacgccggctcgctgtcggccctgctggacccctctcaggggatggggcctagcctgatcggcctcgcgatgggtgacgccggcggttacaaggccgcagacatgtggggtccctcgagtgacccggcatgggagcgcaacgaccctacgcagcagatccccaagctggtcgcaaacaacacccggctatgggtttattgcgggaacggcaccccgaacgagttgggcggtgccaacatacccgccgagttcttggagaacttcgttcgtagcagcaacctgaagttccaggatgcgtacaacgccgcgggcgggcacaacgccgtgttcaacttcccgcccaacggcacgcacagctgggagtactggggcgctcagctcaacgccatgaagggtgacctgcagagttcgttaggcgccggcatgacagagcagcagtggaatttcgcgggtatcgaggccgcggcaagcgcaatccagggaaatgtcacgtccattcattccctccttgacgaggggaagcagtccctgaccaagctcgcagcggcctggggcggtagcggttcggaggcgtaccagggtgtccagcaaaaatgggacgccacggctaccgagctgaacaacgcgctgcagaacctggcgcggacgatcagcgaagccggtcaggcaatggcttcgaccgaaggcaacgtcactgggatgttcgcatag(SEQ ID NO: 25)MFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGNNSPAVYLLDGLRAQDDYNGWDINTPAFEWYYQSGLSIVMPVGGQSSFYSDWYSPACGKAGCQTYKWETFLTSELPQWLSANRAVKPTGSAAIGLSMAGSSAMILAAYHPQQFIYAGSLSALLDPSQGMGPSLIGLAMGDAGGYKAADMWGPSSDPAWERNDPTQQIPKLVANNTRLWVYCGNGTPNELGGANIPAEFLENFVRSSNLKFQDAYNAAGGHNAVFNFPPNGTHSWEYWGAQLNAMKGDLQSSLGAGMTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA (SEQ ID NO: 26) Eatggcgttcagcagacccggcctgcccgtggagtacctgcaggtgcccagccccagcatgggccgggacatcaaagtgcagttccagagcggcggagccaacagccctgccctgtacctgctggacggcctgcgggcccaggacgacttcagcggctgggacatcaacacccccgccacgagtggtacgaccagagcggcctgagcgtggtgatgcccgtgggcggccagagcagatctacagcgactggtatcagcccgcctgcggcaaggccggctgccagacctacaagtgggagaccacctgaccagcgagctgcccggctggctgcaggccaaccggcacgtgaagcccaccggcagcgccgtggtgggcctgagcatggccgccagcagcgccctgaccctggccatctaccacccccagcagttcgtgtacgccggagccatgagcggcctgctggaccccagccaggccatgggccccaccctgatcggcctggccatgggcgacgccggaggctacaaggccagcgacatgtggggccccaaggaggaccccgcctggcagcggaacgaccccctgctgaacgtgggcaagctgatcgccaacaacacccgcgtgtgggtgtactgcggcaacggcaagcccagcgacctgggcggcaacaacctgcccgccaagacctggagggcttcgtgcggaccagcaacatcaagttccaggacgcctacaacgccggaggcggccacaacggcgtgacgacttccccgacagcggcacccacagctgggagtactggggagcccagctgaacgccatgaagcccgacctgcagcgggccctgggcgccacccccaacaccggccctgccccccagggcgctaccgagcagcagtggaacttcgccggcatcgaagctgccgcgagcgccatccaaggcaacgtgaccagcatccacagcctgctggacgagggcaagcagagcctgaccaagctggctgctgcttggggcggatccggaagcgaagcctaccagggcgtgcagcagaagtgggacgccacagccaccgagctgaacaacgccctgcagaacctcgccagaaccatcagcgaggccggacaggctatggccagcacagagggcaatgtgaccggcatgacgccagggccacagtgggcctggtggaggccattggcatcagggagctgaggcagcacgccagcaggtacctggccagagtggaggctggagaggagctgggcgtgaccaacaagggcaggctggtggccagactgatccccgtgcaggctgccgagaggagcagagaggccctgatcgagagcggcgtgctgatccctgccagaaggcctcagaacctgctggacgtgaccgctgagcctgccagaggcaggaagaggaccctgagcgacgtgctgaacgagatgagggacgagcagacaacagccagggacatcatgaacgccggcgtgacctgcgtgggagagcatgaaaccctcaccgccgccgcccaatacatgagggagcacgacatcggcgccctgcccatctgtggagacgacgacaggctgcacggcatgctgaccgacagggacatcgtgatcaagggcctggctgccggcctcgatcctaacaccgctacagccggcgagctggccagagacagcatctactacgtggacgccaacgccagcatccaggagatgctcaacgtgatggaggagcaccaggtgagaagggtgcctgtgatcagcgagcacaggctggtgggcatcgtgaccgaggccgatatcgctaggcacctgcccgagcacgccatcgtgcagttcgtgaaggccatctgcagccccatggctctggccagctcagggaggcatcggaaaccaactacaagcaatgtatctgagccaagattgattcaccggcgcagttcttggaggtggcggaattgccatggctgcccaggcaacagccgctacagatggagagtgggatcaggtggctcgatgtgagtctggtggcaactggtctatcaacactgggaacgggtatcaggcggcttgcaatttactcagagcacttgggctgcccacggagggggtgaatttgctcctagcgcgcagctggcctcccgcgagcagcagatcgctgtgggagagagggtgttggccacacagggaagaggtgcctggcctgtctgtggccgcggactcagtaatgctacccctagggaggtgctgcccgcctcagccgctatggacgctccactggatgctgccgccgtgaatggcgagccagctccgctggcacccccacctgcagaccccgctcccccagtcgagctggcggcaaacgacctgcccgcacctctcggagaaccacttcctgcagcgcctgccgatccagctccacctgctgataggctccccccgctcccgccgatgtagcccctccggtcgagaggctgtgaatgacctgccggcacctctgggcgagcccctcccagccgctccggccgaccctgcccctcctgctgatctggcaccacccgctcctgccgacctcgccccacccgccccagcagacctggctccaccagcgcctgcggatcttgccccgcctgagagctggctgtcaacgatcacctgcgcctcttggagagcccctgcccgctgctccagccgaactcgcaccaccggcagatctggctcccgcctctgccgatcttgcacctcccgcaccggcggacttggcacctccagcaccagcagaactggctccccctgcgccggctgacctggcccctccagcagccgttaatgagcaaaccgcaccaggggaccagccggctacggcaccaggtggaccggtggggctggccaccgacctggagctgcctgagccggatccccaaccagctgatgctcccccacctggcgacgtaactgaggccccagctgaaacgccccaggtcagtaacatcgcttacacaaagaaactgtggcaggcaattagggctcaggacgtgtgtgggaacgacgccctggacagcaggcccaaccgtacgtgatcggtacccccggactcctcaccacagctggagctggcaggcccagagacagatgcgccaggatcgtgtgcaccgtgacatcgagaccgccgtggtggctaccatgacgtggccctgctgggcctgagcaccatcagcagcaaggccgacgacatcgactgggacgccatcgcccagtgtgaatccggcggaaactgggccgccaataccggcaatggcctgtacggcggcctgcagatcagccaggctacctgggactccaacggaggagtgggaagccctgccgctgcttcccctcagcagcagatcgaggtggccgacaacatcatgaagacccaaggccctggcgcctggcctaagtgttccagctgtagccagggcgatgctcctctgggcagcctgacccacatcctgacctttctcgccgccgagacaggcggatgtagcggaagcagggacgac (SEQ ID NO: 27)gctagcaccatggcgttcagcagacccggcctgcccgtggagtacctgcaggtgcccagccccagcatgggccgggacatcaaagtgcagaccagagcggcggagccaacagccctgccctgtacctgctggacggcctgcgggcccaggacgacttcagcggctgggacatcaacacccccgccacgagtggtacgaccagagcggcctgagcgtggtgatgcccgtgggcggccagagcagatctacagcgactggtatcagcccgcctgcggcaaggccggctgccagacctacaagtgggagaccttcctgaccagcgagctgcccggctggctgcaggccaaccggcacgtgaagcccaccggcagcgccgtggtgggcctgagcatggccgccagcagcgccctgaccctggccatctaccacccccagcagttcgtgtacgccggagccatgagcggcctgctggaccccagccaggccatgggccccaccctgatcggcctggccatgggcgacgccggaggctacaaggccagcgacatgtggggccccaaggaggaccccgcctggcagcggaacgaccccctgctgaacgtgggcaagctgatcgccaacaacacccgcgtgtgggtgtactgcggcaacggcaagcccagcgacctgggcggcaacaacctgcccgccaagttcctggagggcttcgtgcggaccagcaacatcaagttccaggacgcctacaacgccggaggcggccacaacggcgtgacgacttccccgacagcggcacccacagctgggagtactggggagcccagctgaacgccatgaagcccgacctgcagcgggccctgggcgccacccccaacaccggccctgccccccagggcgctaccgagcagcagtggaacttcgccggcatcgaagctgccgcgagcgccatccaaggcaacgtgaccagcatccacagcctgctggacgagggcaagcagagcctgaccaagctggctgctgcaggggcggatccggaagcgaagcctaccagggcgtgcagcagaagtgggacgccacagccaccgagctgaacaacgccctgcagaacctcgccagaaccatcagcgaggccggacaggctatggccagcacagagggcaatgtgaccggcatgacgccagggccacagtgggcctggtggaggccattggcatcagggagctgaggcagcacgccagcaggtacctggccagagtggaggctggagaggagctgggcgtgaccaacaagggcaggctggtggccagactgatccccgtgcaggctgccgagaggagcagagaggccctgatcgagagcggcgtgctgatccctgccagaaggcctcagaacctgctggacgtgaccgctgagcctgccagaggcaggaagaggaccctgagcgacgtgctgaacgagatgagggacgagcagacaacagccagggacatcatgaacgccggcgtgacctgcgtgggagagcatgaaaccctcaccgccgccgcccaatacatgagggagcacgacatcggcgccctgcccatctgtggagacgacgacaggctgcacggcatgctgaccgacagggacatcgtgatcaagggcctggctgccggcctcgatcctaacaccgctacagccggcgagctggccagagacagcatctactacgtggacgccaacgccagcatccaggagatgctcaacgtgatggaggagcaccaggtgagaagggtgcctgtgatcagcgagcacaggctggtgggcatcgtgaccgaggccgatatcgctaggcacctgcccgagcacgccatcgtgcagttcgtgaaggccatctgcagccccatggctctggccagctcagggaggcatcggaaaccaactacaagcaatgtatctgagccaagattgattcaccggcgcagttcaggaggtggcggaattgccatggctgcccaggcaacagccgctacagatggagagtgggatcaggtggctcgatgtgagtctggtggcaactggtctatcaacactgggaacgggtatcaggcggcttgcaatttactcagagcacttgggctgcccacggagggggtgaatttgctcctagcgcgcagctggcctcccgcgagcagcagatcgctgtgggagagagggtgttggccacacagggaagaggtgcctggcctgtctgtggccgcggactcagtaatgctacccctagggaggtgctgcccgcctcagccgctatggacgctccactggatgctgccgccgtgaatggcgagccagctccgctggcacccccacctgcagaccccgctcccccagtcgagctggcggcaaacgacctgcccgcacctctcggagaaccacttcctgcagcgcctgccgatccagctccacctgctgataggctccccccgctcccgccgatgtagcccctccggtcgagaggctgtgaatgacctgccggcacctctgggcgagcccctcccagccgctccggccgaccctgcccctcctgctgatctggcaccacccgctcctgccgacctcgccccacccgccccagcagacctggctccaccagcgcctgcggatcttgccccgcctgagagctggctgtcaacgatcttcctgcgcctcttggagagcccctgcccgctgctccagccgaactcgcaccaccggcagatctggctcccgcctctgccgatcttgcacctcccgcaccggcggacttggcacctccagcaccagcagaactggctccccctgcgccggctgacctggcccctccagcagccgttaatgagcaaaccgcaccaggggaccagccggctacggcaccaggtggaccggtggggctggccaccgacctggagctgcctgagccggatccccaaccagctgatgctcccccacctggcgacgtaactgaggccccagctgaaacgccccaggtcagtaacatcgcttacacaaagaaactgtggcaggcaattagggctcaggacgtgtgtgggaacgacgccctggacagcaggcccaaccgtacgtgatcggtacccccggactcctcaccacagctggagctggcaggcccagagacagatgcgccaggatcgtgtgcaccgtgacatcgagaccgccgtggtggctaccatgacgtggccctgctgggcctgagcaccatcagcagcaaggccgacgacatcgactgggacgccatcgcccagtgtgaatccggcggaaactgggccgccaataccggcaatggcctgtacggcggcctgcagatcagccaggctacctgggactccaacggaggagtgggaagccctgccgctgcttcccctcagcagcagatcgaggtggccgacaacatcatgaagacccaaggccctggcgcctggcctaagtgaccagctgtagccagggcgatgctcctctgggcagcctgacccacatcctgacctactcgccgccgagacaggcggatgtagcggaagcagggacgactacccctacgacgtgcccgactacgccgattagtctaga(SEQ ID NO: 28)MAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGATEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFARATVGLVEAIGIRELRQHASRYLARVEAGEELGVTNKGRLVARLIPVQAAERSREALIESGVLIPARRPQNLLDVTAEPARGRKRTLSDVLNEMRDEQTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASSGRHRKPTTSNVSVAKIAFTGAVLGGGGIAMAAQATAATDGEWDQVARCESGGNWSINTGNGYLGGLQFTQSTWAAHGGGEFAPSAQLASREQQIAVGERVLATQGRGAWPVCGRGLSNATPREVLPASAAMDAPLDAAAVNGEPAPLAPPPADPAPPVELAANDLPAPLGEPLPAAPADPAPPADLAPPAPADVAPPVELAVNDLPAPLGEPLPAAPADPAPPADLAPPAPADLAPPAPADLAPPAPADLAPPVELAVNDLPAPLGEPLPAAPAELAPPADLAPASADLAPPAPADLAPPAPAELAPPAPADLAPPAAVNEQTAPGDQPATAPGGPVGLATDLELPEPDPQPADAPPPGDVTEAPAETPQVSNIAYTKKLWQAIRAQDVCGNDALDSLAQPYVIGTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDD (SEQ ID NO: 29)MAFSRPGLPVEYLQVPSPSMGRDIKVQFQSGGANSPALYLLDGLRAQDDFSGWDINTPAFEWYDQSGLSVVMPVGGQSSFYSDWYQPACGKAGCQTYKWETFLTSELPGWLQANRHVKPTGSAVVGLSMAASSALTLAIYHPQQFVYAGAMSGLLDPSQAMGPTLIGLAMGDAGGYKASDMWGPKEDPAWQRNDPLLNVGKLIANNTRVWVYCGNGKPSDLGGNNLPAKFLEGFVRTSNIKFQDAYNAGGGHNGVFDFPDSGTHSWEYWGAQLNAMKPDLQRALGATPNTGPAPQGATEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQKWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFARATVGLVEAIGIRELRQHASRYLARVEAGEELGVTNKGRLVARLIPVQAAERSREALIESGVLIPARRPQNLLDVTAEPARGRKRTLSDVLNEMRDEQTTARDIMNAGVTCVGEHETLTAAAQYMREHDIGALPICGDDDRLHGMLTDRDIVIKGLAAGLDPNTATAGELARDSIYYVDANASIQEMLNVMEEHQVRRVPVISEHRLVGIVTEADIARHLPEHAIVQFVKAICSPMALASSGRHRKPTTSNVSVAKIAFTGAVLGGGGIAMAAQATAATDGEWDQVARCESGGNWSINTGNGYLGGLQFTQSTWAAHGGGEFAPSAQLASREQQIAVGERVLATQGRGAWPVCGRGLSNATPREVLPASAAMDAPLDAAAVNGEPAPLAPPPADPAPPVELAANDLPAPLGEPLPAAPADPAPPADLAPPAPADVAPPVELAVNDLPAPLGEPLPAAPADPAPPADLAPPAPADLAPPAPADLAPPAPADLAPPVELAVNDLPAPLGEPLPAAPAELAPPADLAPASADLAPPAPADLAPPAPAELAPPAPADLAPPAAVNEQTAPGDQPATAPGGPVGLATDLELPEPDPQPADAPPPGDVTEAPAETPQVSNIAYTKKLWQAIRAQDVCGNDALDSLAQPYVIGTPGLLTTAGAGRPRDRCARIVCTVFIETAVVATMFVALLGLSTISSKADDIDWDAIAQCESGGNWAANTGNGLYGGLQISQATWDSNGGVGSPAAASPQQQIEVADNIMKTQGPGAWPKCSSCSQGDAPLGSLTHILTFLAAETGGCSGSRDDYPYDVPDYAD (SEQ ID NO: 30)

Any Mtb antigen, including any Mtb antigen within any of the fusionproteins described herein, can have an amino acid sequence that is 100%,or from 70% to 99.9%, identical to the particular amino acid sequencelisted in Tables 1 and 2. The amino acid sequence of any individual Mtbantigen, including any Mtb antigen within any of the fusion proteinsdescribed herein, can be at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% identical to the particular amino acidsequence listed in Tables 1 and 2. Identity or similarity with respectto an amino acid or nucleotide sequence is defined herein as thepercentage of amino acid residues (or nucleotide residues as the casemay be) in the particular Mtb antigen that are identical (i.e., sameresidue) with the amino acid or nucleotide sequence for the Mtb antigenshown in Tables 1 and 2, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity.Percent sequence identity can be determined by, for example, the Gapprogram (Wisconsin Sequence Analysis Package, Version 8 for Unix,Genetics Computer Group, University Research Park, Madison Wis.), usingdefault settings, which uses the algorithm of Smith and Waterman (Adv.Appl. Math., 1981, 2, 482-489). Any amino acid number calculated as a %identity can be rounded up or down, as the case may be, to the closestwhole number.

Optimal alignment of sequences for comparison can also be conductedusing the Megalign program in the Lasergene suite of bioinformaticssoftware (DNASTAR, Inc., Madison, Wis.), using default parameters. Thisprogram embodies several alignment schemes described in the followingreferences: Dayhoff, M. O. (1978) A model of evolutionary change inproteins—Matrices for detecting distant relationships. In Dayhoff, M. O.(ed.) Atlas of Protein Sequence and Structure, National BiomedicalResearch Foundation, Washington D.C. Vol. 5, Suppl. 3, pp. 345-358; HeinJ. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, Calif.;Higgins, D. G. and Sharp, P. M. (1989) CABIOS 5:151-153; Myers, E. W.and Muller W. (1988) CABIOS 4:11-17; Robinson, E. D. (1971) Comb. Theor11:105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4:406-425; Sneath, P.H. A. and Sokal, R. R. (1973) Numerical Taxonomy—the Principles andPractice of Numerical Taxonomy, Freeman Press, San Francisco, Calif.;Wilbur, W. J. and Lipman, D. J. (1983) Proc. Natl. Acad., Sci. USA80:726-730.

Alternately, optimal alignment of sequences for comparison may beconducted by the local identity algorithm of Smith and Waterman (1981)Add. APL. Math 2:482, by the identity alignment algorithm of Needlemanand Wunsch (1970) J. Mol. Biol. 48:443, by the search for similaritymethods of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or byinspection.

Suitable examples of algorithms for determining percent sequenceidentity and sequence similarity are the BLAST and BLAST 2.0 algorithms,which are described in Altschul et al. (1977) Nucl. Acids Res.25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. BLAST and BLAST 2.0 can be used, for example with theparameters described herein, to determine percent sequence identity forthe polynucleotides and polypeptides of the invention. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. In one illustrative example,cumulative scores can be calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix can be used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915)alignments, (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

In some embodiments, the “percentage of sequence identity” is determinedby comparing two optimally aligned sequences over a window of comparisonof at least 20 positions, wherein the portion of the polynucleotide orpolypeptide sequence in the comparison window may comprise additions ordeletions (i.e., gaps) of 20 percent or less, usually 5 to 15 percent,or 10 to 12 percent, as compared to the reference sequences (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which the identical nucleic acid bases or amino acidresidue occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the reference sequence (i.e., the window size) andmultiplying the results by 100 to yield the percentage of sequenceidentity.

Any Mtb antigen, including any Mtb antigen within any of the fusionproteins described herein, can be fragments of the particular amino acidsequence listed in Table 1. The amino acid sequence of any individualMtb antigen, including any Mtb antigen within any of the fusion proteinsdescribed herein, can be missing consecutive amino acids constituting atleast 20%, at least 15%, at least 10%, at least 5%, at least 4%, atleast 3%, at least 2%, or at least 1%, of the particular amino acidsequence listed in Table 1. The omitted consecutive amino acids may befrom the C-terminus or N-terminus portion of the antigen. Alternately,the omitted consecutive amino acids may be from the internal portion ofthe antigen, thus retaining at least its C-terminus and N-terminus aminoacids of the antigen.

Any Mtb antigen, including any Mtb antigen within any of the fusionproteins described herein, can have one or more amino acid additions,deletions, or substitutions compared to the particular amino acidsequence listed in Table 1. Any individual Mtb antigen, including anyMtb antigen within any of the fusion proteins described herein, can haveat least one, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, or at least twelve amino acid additions,deletions, or substitutions compared to the particular amino acidsequence listed in Table 1. The amino acid additions, deletions, orsubstitutions can take place at any amino acid position within the Mtbantigen.

Where a particular Mtb antigen, including any Mtb antigen within any ofthe fusion proteins described herein, comprises at least one or moresubstitutions, the substituted amino acid(s) can each be, independently,any naturally occurring amino acid or any non-naturally occurring aminoacid. Thus, a particular Mtb antigen may comprise one or more amino acidsubstitutions that are naturally occurring amino acids and/or one ormore amino acid substitutions that are non-naturally occurring aminoacids. Individual amino acid substitutions are selected from any one ofthe following: 1) the set of amino acids with nonpolar sidechains, forexample, Ala, Cys, Ile, Leu, Met, Phe, Pro, Val; 2) the set of aminoacids with negatively charged side chains, for example, Asp, Glu; 3) theset of amino acids with positively charged sidechains, for example, Arg,His, Lys; and 4) the set of amino acids with uncharged polar sidechains,for example, Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, Tyr, towhich are added Cys, Gly, Met and Phe. Substitutions of a member of oneclass with another member of the same class are contemplated herein.Naturally occurring amino acids include, for example, alanine (Ala),arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys),glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His),isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met),phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr),tryptophan (Trp), tyrosine (Tyr), and valine (Val). Non-naturallyoccurring amino acids include, for example, norleucine, omithine,norvaline, homoserine, and other amino acid residue analogues such asthose described in Ellman et al., Meth. Enzym., 1991, 202, 301-336. Togenerate such non-naturally occurring amino acid residues, theprocedures of Noren et al., Science, 1989, 244, 182 and Ellman et al.,supra, can be used. Briefly, these procedures involve chemicallyactivating a suppressor tRNA with a non-naturally occurring amino acidresidue followed by in vitro transcription and translation of the RNA.

The Mtb antigens, including any Mtb antigen within any of the fusionproteins described herein, which are modified as described herein retaintheir ability to elicit an immune response against Mycobacteriumtuberculosis. That is, modification of a particular Mtb antigen,including any Mtb antigen within any of the fusion proteins describedherein, will still allow the resultant Mtb antigen, or fusion proteincomprising the same, to elicit an immune response against Mycobacteriumtuberculosis.

The present disclosure also provides nucleic acid molecules encoding anyof the fusion proteins described herein that comprise at least threeMycobacterium tuberculosis (Mtb) antigens. The nucleic acid moleculesdescribed herein and in Tables 1 and 2 are representative. The specificsequences recited in Table 1 are simply one example of a nucleic acidmolecule that can encode a particular Mtb antigen within a fusionprotein. One skilled in the art having knowledge of the genetic code canroutinely prepare and design a plethora of nucleic acid moleculesencoding the same Mtb antigen. The length and nucleotide content of anyparticular nucleic acid molecule is dictated by the desired amino acidsequence of the encoded Mtb antigen. The nucleic acid molecule sequencesshown in Tables 1 and 2 are DNA, although RNA nucleic acid molecules arealso contemplated.

In some embodiments, the CMV vaccines are attenuated CMV vaccines whichare unable or impaired in their ability to replicate or assemble incells and tissues associated with CMV transmission and disease. Inaddition, the present disclosure includes embodiments that relate to theunique ability of RhCMV to re-infect sero-positive Rhesus Macaques (orHCMV to re-infect sero-positive humans) in spite of the presence of asignificant anti-RhCMV immune response (or anti-HCMV immune response).This inherent property of CMV vectors can be attributed to the extensiverepertoire of immune evasion genes encoded by this virus (Hansen, S. G.,Powers, C. J., Richards, R., Ventura, A. B., Ford, J. C., Siess, D.,Axthelm, M. K., Nelson, J. A., Jarvis, M. A., Picker, L. J., et al.2010. Evasion of CD8+ T cells is critical for superinfection bycytomegalovirus. Science 328:102-106).

Some embodiments address issues of virus shedding and pathogenesis andrelate to two potentially complementary approaches to generate a safeand effective vaccines using the CMV vectors. One approach focuses ondevelopment of CMV vectors that are either completely or conditionallyspread defective or severely restricted in their replication, but thatremain capable of inducing a protective immune response against aheterologous antigen. The second approach focuses on the generation ofreplication competent CMV vectors that are unable to infect cells, suchas epithelial cells, which are a major cell type, along withfibroblasts, in the lung associated with CMV pneumonia. Some embodimentsmay relate to additional safety features into these vectors, including ablock in replication in neural and myeloid cells.

In some embodiments, the HCMV and RhCMV recombinant vectors encodeheterologous antigens that may elicit and maintain high level cellularand/or humoral immune responses specific for the encoded antigen.

Further provided are recombinant RhCMV or HCMV vectors including adeletion in one or more RhCMV or HCMV genes that are important forreplication. In some embodiments, at least one essential or augmentinggene is UL82, UL94, UL32, UL99, UL115 or UL44, or a homolog thereof. Insome embodiments, the recombinant RhCMV or HCMV vectors further includea heterologous antigen, such as a pathogen-specific antigen or a tumorantigen.

For a human CMV (HCMV)/TB vaccine to be safe for all potential subjectsin a general population, including individuals with unsuspected immunecompromise, the CMV vaccine vector needs to be attenuated without losingthe ability to induce protective immunity. CMV can replicate in a widevariety of cells and tissues in the host, including: neurons in thecentral nervous system (CNS), epithelial cells, hepatocytes, lung andkidney. Myeloid and endothelial cells are considered persistent sitesfor CMV in the host. During overt CMV disease in immunocompromisedindividuals, direct infection resulting in destruction of epithelial andendothelial cells in the lung, liver and retina is responsible fordisease in these target organs. During congenital infection, direct CMVinfection of neuronal cells is believed to account for the associatedhearing deficits and mental retardation. Embodiments of the inventionrelate to modulating the ability of CMV to replicate in these criticalcell types in order to increase vector safety without compromisingvaccine efficacy, said attenuated viruses and their use as vaccines.

Some embodiments relate to HCMV as a vector for inducing protectiveimmunity to TB, which is based on the highly innovative hypothesis thata high frequency, effector memory-biased T cell response has distinctadvantages over conventional vaccine generated memory, combined with therecognition that HCMV provides just such a response. This characteristicof HCMV is unique to this virus, even when compared to other persistentviruses such as herpes simplex virus (HSV) and Epstein-Barr virus(Asanuma, H., Sharp, M., Maecker, H. T., Maino, V. C., and Arvin, A. M.2000. Frequencies of memory T cells specific for varicella-zoster virus,herpes simplex virus, and cytomegalovirus by intracellular detection ofcytokine expression. J Infect Dis 181:859-866; Harari, A., Vallelian,F., Meylan, P. R., and Pantaleo, G. 2005. Functional heterogeneity ofmemory CD4+ T cell responses in different conditions of antigen exposureand persistence. J Immunol 174:1037-1045; Harari, A., Enders, F. B.,Cellerai, C., Bart, P. A., and Pantaleo, G. 2009. Distinct profiles ofcytotoxic granules in memory CD8+ T cells correlate with function,differentiation stage, and antigen exposure. J Virol 83:2862-2871;Sylwester, A. W., Mitchell, B. L., Edgar, J. B., Taormina, C., Pelte,C., Ruchti, F., Sleath, P. R., Grabstein, K. H., Hosken, N. A., Kern,F., et al. 2005. Broadly targeted human cytomegalovirus-specific CD4+and CD8+ T cells dominate the memory compartments of exposed subjects. JExp Med 202:673-685).

While the HCMV vaccine may be considered safe, concerns still remainregarding both pathogenicity as well as the ability of the virus tospread to unvaccinated sero-negative individuals. The ability torationally design an HCMV vaccine that is less pathogenic and not shedinto the environment is now available with the advent of technologicalbreakthroughs to clone and genetically manipulate CMV. With a long-termgoal of generating a CMV vaccine vector encoding TB antigens that issafe and unable to spread to other individuals. Embodiments of thisinvention relate to the rational design and use of the latest reversegenetic techniques to generate a CMV-based vector that has a restrictedtropism for cells involved in shedding as well as an altered ability toreplicate in tissues associated with both adult and immunosuppressedpopulations.

In some embodiments, the recombinant RhCMV or HCMV vaccine vector is atropism-restricted vector. In some embodiments, the tropism-restrictivevector lacks genes required for optimal growth in certain cell types orcontains targets for tissue-specific micro-RNAs in genes essential forviral replication or wherein the tropism-restrictive vector has anepithelial, central nervous system (CNS), or macrophage deficienttropism, or a combination thereof.

Some embodiments relate to alteration of the cell-tropism of the CMVvector so as to prevent infection of specific cell types involved inpotential tissue damage and/or shedding into urine or secretions. CMV iscapable of infecting a wide variety of cells in the host, including:epithelial cells in gut, kidney, lung and retina, neuronal cells in theCNS, hepatocytes, as well as endothelial cells and myeloid lineage cellsthat are considered persistent sites of the virus (Dankner, W. M.,McCutchan, J. A., Richman, D. D., Hirata, K., and Spector, S. A. 1990.Localization of human cytomegalovirus in peripheral blood leukocytes byin situ hybridization. J Infect Dis 161:31-36; Einhorn, L., and Ost, A.1984. Cytomegalovirus infection of human blood cells. J Infect Dis149:207-214; Gnann, J. W., Jr., Ahlmen, J., Svalander, C., Olding, L.,Oldstone, M. B., and Nelson, J. A. 1988. Inflammatory cells intransplanted kidneys are infected by human cytomegalovirus. Am J Pathol132:239-248; Howell, C. L., Miller, M. J., and Martin, W. J. 1979.Comparison of rates of virus isolation from leukocyte populationsseparated from blood by conventional and Ficoll-Paque/Macrodex methods.J Clin Microbiol 10:533-537; Myerson, D., Hackman, R. C., Nelson, J. A.,Ward, D. C., and McDougall, J. K. 1984. Widespread presence ofhistologically occult cytomegalovirus. Hum Pathol 15:430-439; Schrier,R. D., Nelson, J. A., and Oldstone, M. B. 1985. Detection of humancytomegalovirus in peripheral blood lymphocytes in a natural infection.Science 230:1048-1051; Sinzger, C., Grefte, A., Plachter, B., Gouw, A.S., The, T. H., and Jahn, G. 1995. Fibroblasts, epithelial cells,endothelial cells and smooth muscle cells are major targets of humancytomegalovirus infection in lung and gastrointestinal tissues. J GenVirol 76:741-750).

HCMV encodes >200 genes and several of the genes that are dispensablefor basic virus replication have been identified as tropism determinantsthat enable the virus to enter and replicate in macrophages, endothelialcells, and epithelial cells. One locus of HCMV genes, UL128-131A, hasbeen shown to be essential for entry into endothelial and epithelialcells (Gerna, G., Percivalle, E., Lilleri, D., Lozza, L., Fornara, C.,Hahn, G., Baldanti, F., and Revello, M. G. 2005. Dendritic-cellinfection by human cytomegalovirus is restricted to strains carryingfunctional UL131-128 genes and mediates efficient viral antigenpresentation to CD8+ T cells. J Gen Virol 86:275-284; Hahn, G., Revello,M. G., Patrone, M., Percivalle, E., Campanini, G., Sarasini, A., Wagner,M., Gallina, A., Milanesi, G., Koszinowski, U., et al. 2004. Humancytomegalovirus UL131-128 genes are indispensable for virus growth inendothelial cells and virus transfer to leukocytes. J Virol78:10023-10033; Wang, D., and Shenk, T. 2005. Human cytomegalovirusUL131 open reading frame is required for epithelial cell tropism. JVirol 79:10330-10338; Wang, D., and Shenk, T. 2005. Humancytomegalovirus virion protein complex required for epithelial andendothelial cell tropism. Proc Natl Acad Sci USA 102:18153-18158;Ryckman, B. J., Rainish, B. L., Chase, M. C., Borton, J. A., Nelson, J.A., Jarvis, M. A., and Johnson, D. C. 2008. Characterization of thehuman cytomegalovirus gH/gL/UL128-131 complex that mediates entry intoepithelial and endothelial cells. J Virol 82:60-70; Ryckman, B. J.,Jarvis, M. A., Drummond, D. D., Nelson, J. A., and Johnson, D. C. 2006.Human cytomegalovirus entry into epithelial and endothelial cellsdepends on genes UL128 to UL150 and occurs by endocytosis and low-pHfusion. J Virol 80:710-722.).

The RhCMV homologues for HCMV UL128 and 130 are inactivated in the RhCMVstrain 68-1. Efficient replication of rhesus cytomegalovirus variants inmultiple rhesus and human cell types. Proc Natl Acad Sci USA105:19950-19955. Interestingly, RhCMV 68-1 still grows in epithelial andendothelial cells (albeit at a reduced rate compared to low passageRhCMV virus with intact UL128/130) (Lilja, A. E., Chang, W. L., Barry,P. A., Becerra, S. P., and Shenk, T. E. 2008. Functional geneticanalysis of rhesus cytomegalovirus: Rh-1 is an epithelial cell tropismfactor. J Virol 82:2170-2181; Rue, C. A., Jarvis, M. A., Knoche, A. J.,Meyers, H. L., DeFilippis, V. R., Hansen, S. G., Wagner, M., Fruh, K.,Anders, D. G., Wong, S. W., et al. 2004. A cyclooxygenase-2 homologueencoded by rhesus cytomegalovirus is a determinant for endothelial celltropism. Journal of Virology 78:12529-12536). Mutational analysis ofRhCMV 68-1 has identified 4 other RhCMV genes (Rh01 (HCMV TLR1), Rh159(HCMV UL148), Rh160 (UL132) and Rh203 (HCMVUS22)) that are also requiredfor epithelial cell tropism (Lilja et al., J Virol, 2008, 82,2170-2181). Some embodiments relate to the mutation of the remainder ofthese epithelial cell tropism genes to highly reduce, if not abrogate,the ability of CMV to infect epithelial cells, thereby preventing itsability to be shed into urine or glandular secretions (i.e., saliva andbreast milk), yet likely not compromise the ability of a CMV vector toinduce a protective immune response to TB.

Moreover, since CMV infection of epithelial cells in the lung and retinaresults in pneumonia and retinitis, respectively, elimination of all theCMV epithelial cell tropism genes may significantly reduce the resultantvector's pathogenic potential. Aspects of the invention relate to thishighly targeted and innovative approach that will significantly enhanceboth the safety of the RhCMV/HCMV vector for use as a TB vaccine, aswell as prevent shedding and the potential spread of the vaccine vectorinto the unvaccinated population.

Further embodiments relate to exploiting the tissue-specific expressionof cellular microRNAs (miRNAs) to attenuate the virus in tissuesassociated with disease in adult and congenital infection. EndogenousmicroRNA can be broadly exploited to regulate transgene expressionaccording to tissue, lineage and differentiation state. (Barnes et al.,Cell Host Microbe, 2008, 4, 239-248; Lee et al., Clin. Cancer Res.,2009, 15, 5126-5135; Perez et al., Nat. Biotechnol., 2009, 27, 572-576).

Tissue specific expression of miRNAs has been exploited to generate anattenuated polio vaccine through the introduction of multiple miRNAtarget sequences of miR-124 that is specifically expressed in the CNSinto the 3′UTR of the poliovirus genome (Barnes et al., supra). Additionof the miR-124 target sequences to the poliovirus genome was observed tosignificantly attenuate virus infection in mice. Similarly, multipletarget sequences of miR-93 that is ubiquitously expressed in allmammalian but not avian tissues were added to the nucleoprotein gene ofinfluenza resulting in a species-restricted influenza mutant that wasable to grow in chicken eggs but not in mice (Perez et al., supra).

Some embodiments relate to this attenuation approach being effective forlarger viruses, such as murine CMV (MCMV). Unlike the small RNA viruses,CMV encodes over 200 genes of which approximately 50% are essential andnecessary for replication or encode structural proteins of the virus.One of these essential MCMV genes is the immediate early (IE) 3 gene(the mouse correlate of IE2 in HCMV or RhCMV) that encodes atranscriptional regulatory protein necessary for subsequent activationof early and late genes in the virus. Deletion of this gene completelyblocks viral replication in cells and mouse tissues (Angulo et al., J.Virol., 2000, 74, 11129-11136). It is described herein that introductionof target sequences of tissue-specific miRNAs into the 3′UTR of thisgene would attenuate viral replication in these cells.

In further embodiments, the CMV vector may comprise one or more microRNArecognition elements (MREs). A mature microRNA (interchangeably termedan miRNA or miR) is typically an 18-25 nucleotide non-coding RNA thatregulates expression of an mRNA operably linked to an MRE withspecificity for the miRNA. An MRE can be any sequence that base pairswith and interacts with the miRNA somewhere on the mRNA transcript.Often, the MRE is present in the 3′ untranslated region (UTR) of themRNA, but it can also be present in the coding sequence or in the5′-UTR. MRE's are not necessarily perfect complements to miRNAs, usuallyhaving only a few bases of complementarity to the miRNA and oftencontaining one or more mismatches within those bases of complementarity.An MRE, therefore, can be any sequence capable of being bound by anmiRNA sufficiently that the translation of a gene to which the MRE isoperably linked (such as a CMV gene that is essential or augmenting forgrowth in vivo) is repressed by an miRNA silencing mechanism such as theRISC.

In some examples, a microRNA recognition element (MRE) is operablylinked to a CMV gene that is essential or augmenting for growth in vivo.In other examples, the MRE silences expression in the presence of amiRNA that is expressed in cells of the myeloid lineage. Such miRNAinclude, but are not limited to, miR-142-3p, miR-223, miR-27a, miR-652,miR-155, miR146a, miR-132, miR-21, or miR-125 (Brown et al., Nat.Biotechnol., 2007, 25, 1457-1467). Myeloid lineage cells have been shownto represent a reservoir of latent virus, and are thought to harbor anddisseminate virus throughout the host (Jarvis and Nelson, Front Biosci.,2002, 7, d1575-1582).

Further studies with MCMV (Snyder et al., C. M., Allan, J. E., Bonnett,E. L., Doom, C. M., and Hill, A. B. Cross-presentation of aspread-defective MCMV is sufficient to prime the majority ofvirus-specific CD8+ T cells. PLoS One 5:e9681) indicate thatcross-priming is the primary mechanism by which CMV-encoded proteinsprime the immune response, replication in myeloid dendritic cells mayhave a surprisingly minimal impact on CMV immunogenicity.

Bacterial artificial chromosome (BAC)-based technology is used togenerate a recombinant MCMV virus that contained four repeated targetsequences (four 21mers) with exact complementarity to the cellularmiRNA, miR-142-3p, within the 3′UTR of the essential viral gene IE3(IE3-142). To confirm the extent to which miR-142-3p expression couldrepress 1E3-142 replication, virus growth assays are performed in themacrophage cell line, IC-21. RT-PCR analysis confirmed that IC-21 cellsexpress high levels of miR-142-3p making the cell line suitable to testthe effectiveness of the strategy. Preliminary experiments confirmed theutility of the approach for cell-type specific attenuation of CMV.Although IE3-142 replicated to wild type levels in fibroblasts, growthwas completely blocked in IC-21 macrophage cells. A control virus,IE3-015, which contains only vector sequence within the IE3 insertionsite, replicates to wild-type levels in IC-21 cells. RT-PCR analysisindicates that IE3 expression was completely abrogated followinginfection of IC-21 cells, but not following infection of fibroblastcells (lacking miR-142-3p expression) indicating that disruption of 1E3expression is not due to insertion of the target sequence.

Some embodiments relate to strategy to attenuate CMV based on theshowing that viruses can be attenuated for tissue-specific growth byusing miRNA target sequences and the attenuation of MCMV in myeloidcells through the targeting of cell specific miRNAs to essential viralgenes. Since the CNS is a major target for CMV pathogenesis in bothcongenital and adult disease, HCMV/TB vaccines are generated thatcontain target sequences of highly conserved miRNAs specificallyexpressed by neurons fused to essential CMV genes to prevent replicationin the CNS. Target sequences of the myeloid miRNA miR-124 to preventreplication and dissemination of the CMV vector in this cell type arealso used. Together, these attenuated viruses will provide a furtherlevel of safety that will enable the use of this vaccine in all humantarget populations.

Also disclosed herein are recombinant CMV vectors, such as RhCMV andHCMV vectors, having a deletion in one or more genes that are essentialfor or augment CMV replication, dissemination or spreading. Thus, thesevectors are referred to as “replication-deficient” CMV vectors. As usedherein, “replication-deficient” encompasses CMV vectors that are unableto undergo any replication in a host cell, or have a significantlyreduced ability to undergo viral replication. In some examples, thereplication-deficient CMV vectors are able to replicate, but are unableto disseminate since they are incapable of infection neighboring cells.In some examples, the replication-deficient CMV vectors are able toreplicate, but are unable to spread since they are not released frominfected hosts.

CMV essential and augmenting genes are well known in the art (see, forexample, Dunn et al., Proc. Natl. Acad. Sci. USA, 2003, 100,14223-14228; and Dong et al., Proc. Natl. Acad. Sci. USA, 2003, 100,12396-12401), and are described herein. In some embodiments, therecombinant RhCMV or HCMV vector includes a deletion in one gene that isessential for or augments virus replication, dissemination or spreading.In other embodiments, the recombinant RhCMV or HCMV vector includes adeletion in multiple (such as, but not limited to, two, three or four)genes essential for or augmenting CMV replication, dissemination orspreading. The deletion need not be a deletion of the entire openreading frame of the gene, but includes any deletion that eliminatesexpression of functional protein.

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorcomprises a deletion in a RhCMV or HCMV gene that is essential forreplication within a host, dissemination within a host, or spreadingfrom host to host. In some embodiments, the essential gene is UL82(encoding pp71), UL94 (encoding the UL94 protein), UL32 (encodingpp150), UL99 (encoding pp28), UL115 (encoding gL) and UL44 (encodingp52), or a homolog thereof.

Replication-deficient RhCMV and HCMV vectors disclosed herein caninclude a nucleic acid sequence encoding a heterologous antigen, such asa pathogen-specific antigen. As disclosed for other recombinant RhCMVand HCMV vectors described herein, replication-deficient RhCMV and HCMVvectors can be used to elicit an immune response in a subject againstthe encoded heterologous antigen.

A recombinant RhCMV vector having a deletion in gene UL82 (which encodesthe pp71 protein) is severely impaired in its ability to grow in vitroand to spread in vivo, but still elicits a robust T cell immune responseagainst CMV (U.S. Pat. No. 9,249,427). Thus, it is contemplated hereinto use such a replication-deficient vector as a vaccine against CMVitself.

In some embodiments, the recombinant RhCMV or HCMV vaccine vector has adeletion in a gene region non-essential for growth in vivo. In someembodiments, the gene region is selected from the group consisting ofthe RL11 family, the pp65 family, the US12 family, and the US28 family.In some embodiments, the RhCMV gene region is selected from the groupconsisting of Rh13-Rh29, Rh111-Rh112, Rh191-Rh202, and Rh214-Rh220. Insome embodiments, the RhCMV gene region is selected from the groupconsisting of Rh13.1, Rh19, Rh20, Rh23, Rh24, Rh112, Rh190, Rh192,Rh196, Rh198, Rh199, Rh200, Rh201, Rh202, and Rh220. In someembodiments, the HCMV gene region is selected from the group consistingof RL11, UL6, UL7, UL9, UL11, UL83 (pp65), US12, US13, US14, US17, US18,US19, US20, US21, and UL28.

In some embodiments, the recombinant RhCMV or HCMV vector comprises adeletion in a RhCMV or HCMV gene that is essential for or augmentsreplication. CMV essential genes and augmenting have been well describedin the art (see, for example, Dunn et al., supra; and Dong et al.,supra). Essential CMV genes include, but are not limited to, UL32, UL34,UL37, UL44, UL46, UL48, UL48.5, UL49, UL50, UL51, UL52, UL53, UL54,UL55, UL56, UL57, UL60, UL61, UL70, UL71, UL73, UL75, UL76, UL77, UL79,UL80, UL82, UL84, UL85, UL86, UL87, UL89, UL90, UL91, UL92, UL93, UL94,UL95, UL96, UL98, UL99, UL100, UL102, UL104, UL105, UL115 and UL122. Insome embodiments, the CMV essential or augmenting gene is UL82, UL94,UL32, UL99, UL115 or UL44, or a homolog thereof (i.e., the homologousgene in RhCMV). Other essential or augmenting genes are known in the artand are described herein. In particular examples, the essential gene isUL82, or a homolog thereof. In some embodiments, the recombinant RhCMVand HCMV vectors do not include a heterologous antigen. In otherembodiments, the recombinant RhCMV or HCMV vector having a deletion inan essential or augmenting gene includes a nucleic acid sequenceencoding a heterologous antigen, such as a pathogen-specific antigen ora tumor antigen. Compositions comprising recombinant RhCMV or HCMVvectors and a pharmaceutically acceptable carrier also are provided.Such vectors and compositions can be used, for example, in a method oftreating a subject with an infectious disease, or at risk of becominginfected with an infectious disease. CMV vectors having a deletion of atleast one important gene are generally attenuated and thus can be usedas vaccines for the treatment or prevention of CMV (in which case, therecombinant vector does not encode a heterologous antigen).

In some embodiments, the recombinant RhCMV or HCMV vectors comprise asuicide or safety means to prevent further replication of the virus. Forexample, the recombinant CMV vectors can include LoxP sites flanking anessential gene or region of the RhCMV or HCMV genome (essential CMVgenes are listed above and are known in the art), as well as the codingsequence for Cre-recombinase. Cre-recombinase is generally under thecontrol of an inducible promoter to regulate expression of Cre, therebycontrolling removal of the essential gene and inhibition of viralreplication. In particular examples, Cre is a Tet-regulated Cre andexpression of Cre is controlled by the presence of Dox.

The present disclosure also relates to a method of a CMV vector capableof repeatedly infecting an organism which may comprise (a) constructinga vector containing and over-expressing at least one cytomegalovirus(CMV) glycoprotein, wherein the glycoprotein is US2, US3, US6 or US11,and (b) administering the vector repeatedly into the animal or human.Where superinfectivity is desired, any CMV vector, may express one ormore of the HCMV glycoproteins US2, US3, US6 and US11 (or the RhCMVhomologues Rh182, Rh184, Rh185, Rh189).

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorfurther comprises a second nucleic acid sequence encoding US2, US3, orUS6, or a homolog thereof, wherein the vector does not encode afunctional US11. In some embodiments, the second nucleic acid sequenceencodes US2, US3, and US6. In some embodiments, the nucleic acidencoding a US11 open reading frame is deleted. In some embodiments, therecombinant RhCMV or HCMV vaccine vector further comprises a thirdnucleic acid sequence encoding US11, and wherein the nucleic acidsequence encoding US11 comprises a point mutation, a frameshiftmutation, and/or a deletion of one or more nucleotides of the nucleicacid sequence encoding US11.

In some embodiments, the glycoproteins within the US2 to US11 region ofRhCMV or HCMV are deleted from the vector. In some embodiments, therecombinant RhCMV or HCMV vaccine vector lacks the transactivator pp71.In some embodiments, the recombinant RhCMV or HCMV vaccine vector lacksthe tegument protein pp65.

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorfurther comprises a nucleic acid sequence that encodes UL128 or anortholog thereof, and another nucleic acid sequence that encodes UL131or an ortholog thereof, wherein the vector does not express an activeUL130 protein.

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorfurther comprises a nucleic acid sequence that encodes UL130 or anortholog thereof, and another nucleic acid sequence that encodes UL131or an ortholog thereof, wherein the vector does not express an activeUL128 protein.

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorcomprises a mutation in UL128 or UL130 selected from a point mutation, aframeshift mutation, and a deletion of all or less than all of UL128 orUL130.

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorfurther comprises an antisense sequence or an RNAi sequence thatinhibits the expression of UL128 or UL130 or both.

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorcomprises a deletion or modification of US2, US3, US4, US5, US6, US11,or UL97, or a homolog thereof.

In some embodiments, the recombinant RhCMV or HCMV vaccine vectorcomprises a deletion of Rh158-166 or a homolog thereof.

In some embodiments where repeated infection of a CMV vector is desired,the CMV vector may express one or more of the glycoproteins US2, US3,US6 and US11. In a particularly advantageous embodiment, the vectorexpresses glycoproteins US2, US3, US6 and US11. More advantageously, thevector contains and expresses all of the glycoproteins within the US2 toUS11 region of CMV. In an advantageous embodiment, the one or more ofthe glycoproteins US2, US3, US6 and US11 may include, but not limitedto, the glycoproteins of U.S. Pat. Nos. 7,892,564; 7,749,745; 7,364,893;6,953,661; 6,913,751; 6,740,324; 6,613,892; 6,410,033; 6,140,114;6,103,531; 6,033,671; 5,908,780; 5,906,935; 5,874,279; 5,853,733;5,846,806; 5,843,458; 5,837,532; 5,804,372; 5,753,476; 5,741,696;5,731,188; 5,720,957; 5,676,952; 5,599,544; 5,593,873 and 5,334,498.

Disclosed herein are human or animal CMV vectors comprising a nucleicacid sequence that encodes a heterologous protein antigen and a nucleicacid sequence that encodes an active UL131 protein. In one example, theCMV vector comprises a nucleic acid sequence that expresses an activeUL128 protein but does not express an active UL130 protein. In anotherexample, the CMV vector encodes an active UL130 protein but does notexpress an active UL128 protein.

In some examples, the CMV vector does not express an active UL128 orUL130 protein due to the presence of a deleterious mutation in thenucleic acid sequence encoding UL128 or UL130 or their orthologous genesin animal CMVs. The mutation may be any deleterious mutation thatresults in a lack of expression of active UL128 or UL130 protein. Suchmutations can include point mutations, frameshift mutations, deletionsof less than all of the sequence that encodes the protein (truncationmutations), or deletions of all of the nucleic acid sequence thatencodes the protein, or any other mutations.

In further examples, the CMV vector does not express an active UL128 orUL130 protein due to the presence of a nucleic acid sequence in thevector that comprises an antisense or RNAi sequence (siRNA or miRNA)that inhibits the expression of the UL128 or UL130 protein.

Also disclosed herein are methods of generating CD8+ T cell responses toheterologous antigens in a subject. The methods involve administering aneffective amount of a CMV vector to the subject. The CMV vector ischaracterized by having a nucleic acid sequence that encodes aheterologous antigen and a nucleic acid sequence that encodes an activeUL131 protein. The CMV vector is further characterized by not encodingan active UL128 protein or an active UL130 protein or neither an activeUL128 or active UL130 protein. The CD8+ T cell response is furthercharacterized by having at least 10% of the CD8+ T cells directedagainst epitopes presented by MHC class II. In further examples, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, ormore than 60% of the CD8+ T cells are directed against epitopespresented by MHC class II.

In further examples, the methods involve administering an effectiveamount of a second CMV vector, the second CMV vector comprising anucleic acid sequence that encodes a heterologous antigen to thesubject. This second vector can be any CMV vector, including a CMVvector with an active UL128 and an active UL130 protein. The second CMVvector may comprise additional deletions known in the art to providedifferent immune responses such as a US11 deletion or any otherdeletion. The second heterologous antigen can be any heterologousantigen, including a heterologous antigen identical to the heterologousantigen in the first CMV vector. The second CMV vector can beadministered at any time relative to the administration of the first CMVvector including before, concurrently with, or after the administrationof the first CMV vector. This includes administration of the secondvector any number of months, days, hours, minutes or seconds before orafter the first vector. In preferred embodiments of the presentinvention viral vectors are used. Advantageously, the vector is a CMVvector, lacking at least the glycoprotein UL128 or a CMV vector lackingat least the glycoprotein UL130. Each CMV vector also expresses theglycoprotein UL131.

Suitable dosages of the CMV vectors in the immunogenic compositions canbe readily determined by those of skill in the art. For example, thedosage of the CMV vectors can vary depending on the route ofadministration and the size of the subject. Suitable doses can bedetermined by those of skill in the art, for example by measuring theimmune response of a subject, such as a laboratory animal, usingconventional immunological techniques, and adjusting the dosages asappropriate. Such techniques for measuring the immune response of thesubject include but are not limited to, chromium release assays,tetramer binding assays, IFN-.gamma. ELISPOT assays, IL-2 ELISPOTassays, intracellular cytokine assays, and other immunological detectionassays, e.g., as detailed in the text “Antibodies: A Laboratory Manual”by Ed Harlow and David Lane.

In some embodiments, the recombinant RhCMV vaccine vector is Rh68-1 orRh68-1.2. During in vitro culture on fibroblasts, the Rh68-1 CMV vectorlost the ability to express gene products from the Rh13, Rh60, Rh157.5,and Rh157.6 open reading frames. The HCMV orthologs of these genes areRL11, UL36, UL128, and UL130, respectively. The Rh68-1.2 vector hadexpression of Rh60, Rh157.5, and Rh157.6 restored through recombinantDNA techniques (Lilja and Shenk, Proc. Natl. Acad. Sci. USA, 2008, 105,19950-19955). The Rh68-1 CMV vector, but not the Rh68-1.2 CMV vector,primes surprisingly high number of CD8⁺ T cells restricted by MHC-E.

In some embodiments, the CMV vectors can comprise regulatory elementsfor gene expression of the coding sequences of the nucleic acid. Theregulatory elements can be a promoter, an enhancer an initiation codon,a stop codon, a polyadenylation signal, additional restriction enzymesites, multiple cloning sites, or other coding segments, and the like.In some embodiments, the CMV vector can comprise heterologous nucleicacid encoding an Mtb antigen and can further comprise an initiationcodon, which is upstream of the antigen coding sequence, and a stopcodon, which is downstream of the antigen coding sequence. Theinitiation and termination codon are in frame with the antigen codingsequence.

In some embodiments, expression of the Mtb antigen is driven by anantigen-coding sequence in operable association with a promoter selectedfrom the group consisting of a constitutive CMV promoter, an immediateearly CMV promoter, an early CMV promoter, and a late CMV promoter. Insome embodiments, the promoter is selected from the group consisting ofEF1-alpha, UL82, MIE, pp65, and gH.

The CMV vector can also comprise a polyadenylation signal, which can bedownstream of the antigen coding sequence. The polyadenylation signalcan be a SV40 polyadenylation signal, LTR polyadenylation signal, CMVpolyadeylation signal, bovine growth hormone (bGH) polyadenylationsignal, human growth hormone (hGH) polyadenylation signal, or humanβ-globin polyadenylation signal. The SV40 polyadenylation signal can bea polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego,Calif.).

The CMV vector can also comprise an enhancer. In some embodiments, theenhancer can be necessary for DNA expression. The enhancer can be humanactin, human myosin, human hemoglobin, human muscle creatine or a viralenhancer such as one from CMV, RSV or EBV. Polynucleotide functionenhances are described in U.S. Pat. Nos. 5,593,972, 5,962,428, andWO94/016737, the contents of each are incorporated herein by reference.

The CMV vector can also comprise a mammalian origin of replication tomaintain the vector extrachromosomally and produce multiple copies ofthe vector in a cell. The CMV vector can comprise the Epstein Barr virusorigin of replication and nuclear antigen EBNA-1 coding region, whichcan produce high copy episomal replication without integration. The CMVvector can contain certain elements of the pVAX1 or a pVax1 variant. Thevariant pVax1 plasmid is a 2998 basepair variant of the backbone vectorplasmid pVAX1 (Invitrogen, Carlsbad Calif.). The CMV promoter is locatedat bases 137-724. The T7 promoter/priming site is at bases 664-683.Multiple cloning sites are at bases 696-811. Bovine GH polyadenylationsignal is at bases 829-1053. The Kanamycin resistance gene is at bases1226-2020. The pUC origin is at bases 2320-2993.

The CMV vector can also comprise a regulatory sequence, which can bewell suited for gene expression in a mammalian or human cell into whichthe vector is administered. The consensus coding sequence can comprise acodon, which can allow more efficient transcription of the codingsequence in the host cell.

The present disclosure also provides host cells comprising any of thenucleic acid molecules or CMV vectors disclosed herein. The host cellscan be used, for example, to express the Mtb antigens, or fragments ofthereof. The Mtb antigens, or fragments thereof, can also be expressedin cells in vivo. The host cell that is transformed (for example,transfected) to produce the Mtb antigens, or fragments of thereof can bean immortalised mammalian cell line, such as those of lymphoid origin(for example, a myeloma, hybridoma, trioma or quadroma cell line). Thehost cell can also include normal lymphoid cells, such as B-cells, thathave been immortalized by transformation with a virus (for example, theEpstein-Barr virus).

In some embodiments, the host cells include, but are not limited to:bacterial cells, such as E. coli, Caulobacter crescentus, Streptomycesspecies, and Salmonella typhimurium; yeast cells, such as Saccharomycescerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Pichiamethanolica; insect cell lines, such as those from Spodoptera frugiperda(for example, Sf9 and Sf21 cell lines, and expresSF™ cells (ProteinSciences Corp., Meriden, Conn., USA)), Drosophila S2 cells, andTrichoplusia in High Five® Cells (Invitrogen, Carlsbad, Calif., USA);and mammalian cells, such as COS1 and COS7 cells, Chinese hamster ovary(CHO) cells, NS0 myeloma cells, NIH 3T3 cells, 293 cells, Procell92S,perC6, HEPG2 cells, HeLa cells, L cells, HeLa, MDCK, HEK293, WI38,murine ES cell lines (for example, from strains 129/SV, C57/BL6, DBA-1,129/SVJ), K562, Jurkat cells, and BW5147. Other useful mammalian celllines are well known and readily available from the American TypeCulture Collection (“ATCC”) (Manassas, Va., USA) and the NationalInstitute of General Medical Sciences (NIGMS) Human Genetic CellRepository at the Coriell Cell Repositories (Camden, N.J., USA). In someembodiments, the cell is a recombinant BCG. These cell types are onlyrepresentative and are not meant to be an exhaustive list.

Among other considerations, some of which are described above, a hostcell strain may be chosen for its ability to process the expressed Mtbantigens, or fragment thereof, in the desired fashion.Post-translational modifications of the polypeptide include, but are notlimited to, glycosylation, acetylation, carboxylation, phosphorylation,lipidation, and acylation, and it is an aspect of the present disclosureto provide Mtb antigens thereof with one or more of thesepost-translational modifications.

In some embodiments, the recombinant BCG has been genetically engineeredto express a functional endosomalytic protein that is bioactive at pHvalues near neutrality (e.g. about pH 6-8 or about 6.5 to 7.5). Theendosomalytic protein is active within Mycobacteria-containingendosomes, which typically have an internal pH near neutrality. Theactivity of the endosomalytic protein produced by the rBCG results indisruption of the endosome, permitting the rBCG to escape from theendosome and into the cytoplasm of the cell. In some embodiments, theendosomalytic protein that is introduced into the rBCG by geneticengineering is Perfringolysin O (PfoA) from Clostridium perfringens or amutant thereof, such as PfoA_(G137Q), as described in WO 2007/058663,which is incorporated herein by reference in its entirety.

In some embodiments, the Mycobacteria are attenuated, as exemplified byBCG. However, those of skill in the art will recognize that otherattenuated and nonattenuated Mycobacteria exist which would also besuitable for use herein. Examples of additional types of Mycobacteriainclude, but are not limited to, M. tuberculosis strain CDC1551, M.tuberculosis strain Beijing, M. tuberculosis strain H37Ra (ATCC#:25177), M. tuberculosis strain H37Rv (ATCC #:25618), M. bovis (ATCC#:19211 and 27291), M. fortuitum (ATCC #:15073), M. smegmatis (ATCC#:12051 and 12549), M. intracellulare (ATCC #:35772 and 13209), M.kansasii (ATCC #:21982 and 35775) M. avium (ATCC #:19421 and 25291), M.gallinarum (ATCC #:19711), M. vaccae (ATCC #:15483 and 23024), M. leprae(ATCC #:), M. marinarum (ATCC #:11566 and 11567), and M. microtti (ATCC#:11152).

Examples of attenuated Mycobacterium strains include, but are notrestricted To, M. tuberculosis pantothenate auxotroph strain, M.tuberculosis rpoV mutant strain, M. tuberculosis leucine auxotrophstrain, BCG Danish strain (ATCC #35733), BCG Japanese strain (ATCC#35737), BCG Chicago strain (ATCC #27289), BCG Copenhagen strain (ATCC#: 27290), BCG Pasteur strain (ATCC #: 35734), BCG Glaxo strain (ATCC #:35741), BCG Connaught strain (ATCC #35745), BCG Montreal (ATCC #35746),BCG1331 strain, BCG Tokyo strain, BCG Moreau strain, BCG-Pasteur Aeras,and BCG Moscow strain.

The present disclosure also provides pharmaceutical compositionscomprising any one or more of the recombinant RhCMV or HCMV vaccinevectors described herein and a pharmaceutically acceptable carrier.

In some embodiments, the Mtb antigen, or fragment thereof, is labeledwith a detectable marker. Detectable markers include, but are notlimited to, radioactive isotopes (such as P³² and S³⁵), enzymes (such ashorseradish peroxidase, chloramphenicol acetyltransferase (CAT),β-galactosidase (β-gal), and the like), fluorochromes, chromophores,colloidal gold, dyes, and biotin. The labeled Mtb antigens, or fragmentsthereof, can be used to carry out diagnostic procedures in a variety ofcell or tissue types. For imaging procedures, in vitro or in vivo, theMtb antigens can be labeled with additional agents, such as NMRcontrasting agents, X-ray contrasting agents, or quantum dots. Methodsfor attaching a detectable agent to polypeptides are known in the art.The Mtb antigens can also be attached to an insoluble support (such as abead, a glass or plastic slide, or the like).

In some embodiments, the Mtb antigens, or fragment thereof, can beconjugated to a therapeutic agent including, but not limited to,radioisotopes (such as ¹¹¹In or ⁹⁰Y), toxins (such as tetanus toxoid orricin), toxoids, and chemotherapeutic agents.

In some embodiments, the Mtb antigens, or fragments thereof, can beconjugated to an imaging agent. Imaging agents include, for example, alabeling moiety (such as biotin, fluorescent moieties, radioactivemoieties, histidine tag or other peptide tags) for easy isolation ordetection.

The present disclosure also provides compositions comprising any one ormore of the fusion proteins, Mtb antigens, nucleic acid moleculesencoding Mtb antigens, including fusion proteins thereof, cells, and/orCMV vectors and a pharmaceutically acceptable carrier useful in, forexample, vaccines.

In some embodiments, liquid formulations of a pharmaceutical compositionfor oral administration prepared in water or other aqueous vehicles cancontain various suspending agents such as methylcellulose, alginates,tragacanth, pectin, kelgin, carrageenan, acacia, polyvinylpyrrolidone,and polyvinyl alcohol. Liquid formulations of pharmaceuticalcompositions can also include solutions, emulsions, syrups and elixirscontaining, together with the active compound(s), wetting agents,sweeteners, and coloring and flavoring agents. Various liquid and powderformulations of the pharmaceutical compositions can be prepared byconventional methods for inhalation into the lungs of the mammal to betreated.

In some embodiments, liquid formulations of a pharmaceutical compositionfor injection can comprise various carriers such as vegetable oils,dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate,isopropyl myristate, ethanol, polyols such as, for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like. In someembodiments, the composition includes a citrate/sucrose/tween carrier.For intravenous injections, water soluble versions of the compositionscan be administered by the drip method, whereby a pharmaceuticalformulation containing the antifungal agent and a physiologicallyacceptable excipient is infused. Physiologically acceptable excipientscan include, for example, 5% dextrose, 0.9% saline, Ringer's solution orother suitable excipients. A suitable insoluble form of the compositioncan be prepared and administered as a suspension in an aqueous base or apharmaceutically acceptable oil base, such as an ester of a long chainfatty acid such as, for example, ethyl oleate.

The compositions can be, for example, injectable solutions, aqueoussuspensions or solutions, non-aqueous suspensions or solutions, solidand liquid oral formulations, salves, gels, ointments, intradermalpatches, creams, aerosols, lotions, tablets, capsules, sustained releaseformulations, and the like. In some embodiments, for topicalapplications, the pharmaceutical compositions can be formulated in asuitable ointment. In some embodiments, a topical semi-solid ointmentformulation typically comprises a concentration of the active ingredientfrom about 1 to 20%, or from 5 to 10%, in a carrier, such as apharmaceutical cream base. Some examples of formulations of acomposition for topical use include, but are not limited to, drops,tinctures, lotions, creams, solutions, and ointments containing theactive ingredient and various supports and vehicles.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicroparticles such as polylactide, polyglycolide, or copolymer forenhanced adjuvant effect (see Langer, Science, 1990, 249, 1527 andHanes, Advanced Drug Delivery Reviews, 1997, 28, 97). A sterileinjectable preparation such as, for example, a sterile injectableaqueous or oleaginous suspension can also be prepared. This suspensionmay be formulated according to techniques known in the art usingsuitable dispersing, wetting, and suspending agents. In someembodiments, the pharmaceutical composition can be delivered in amicroencapsulation device so as to reduce or prevent a host immuneresponse against the protein.

A suitable dose is an amount of a compound that, when administered asdescribed above, is capable of promoting an anti-TB immune response, andis preferably at least 10-50% above the basal (i.e., untreated) level.Such response can be monitored, for example, by measuring the anti-Tcell responses in a patient. Such vaccines should also be capable ofcausing an immune response that leads to an improved clinical outcome(e.g., more frequent remissions, complete or partial or longerdisease-free survival) in vaccinated patients as compared tonon-vaccinated patients. In general, for pharmaceutical compositions andvaccines comprising one or more polypeptides, the amount of eachpolypeptide sought in a dose ranges from about 25 mcg to 5 mg per kg ofhost. Suitable dose sizes will vary with the size of the patient, butwill typically range from about 0.1 mL to about 5 mL.

In general, an appropriate dosage and treatment regimen provides theactive compound(s) in an amount sufficient to provide therapeutic and/orprophylactic benefit. Such a response can be monitored by establishingan improved clinical outcome (e.g. more frequent remissions, complete orpartial, or longer disease-free survival) in treated patients ascompared to non-treated patients. Increases in preexisting immuneresponses to a TB protein may correlate with an improved clinicaloutcome. Such immune responses may generally be evaluated using standardproliferation, cytotoxicity or cytokine assays, which may be performedusing samples obtained from a patient before and after treatment.

In some embodiments, the compositions comprise about 1 nanogram to about10 mg of nucleic acid. In some embodiments, the compositionscomprise: 1) at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95 or 100 nanograms, or at least 1, 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105,110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245,250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315,320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385,390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455,460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620, 625,630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695,700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765,770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835,840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895. 900, 905,910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975,980, 985, 990, 995 or 1000 micrograms, or at least 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg or more; and 2)up to and including 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95 or 100 nanograms, or up to and including 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240,245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310,315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380,385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450,455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 605, 610, 615, 620,625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690,695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760,765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830,835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895. 900,905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970,975, 980, 985, 990, 995, or 1000 micrograms, or up to and including 1.5,2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg.

In some embodiments, the compositions comprise about 5 nanograms toabout 10 mg of nucleic acid molecule. In some embodiments, thecompositions comprise about 25 nanograms to about 5 mg of nucleic acidmolecule. In some embodiments, the compositions contain about 50nanograms to about 1 mg of nucleic acid molecule. In some embodiments,the compositions contain about 0.1 to about 500 micrograms of nucleicacid molecule. In some embodiments, the compositions contain about 1 toabout 350 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 5 to about 250 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about to about200 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 15 to about 150 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 20 toabout 100 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 25 to about 75 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 30 toabout 50 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 35 to about 40 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 100 toabout 200 micrograms of nucleic acid molecule. In some embodiments, thecompositions comprise about 10 to about 100 micrograms of nucleic acidmolecule. In some embodiments, the compositions comprise about 20 toabout 80 micrograms of nucleic acid molecule. In some embodiments, thecompositions comprise about 25 to about 60 micrograms of nucleic acidmolecule. In some embodiments, the compositions comprise about 30nanograms to about 50 micrograms of nucleic acid molecule. In someembodiments, the compositions comprise about 35 nanograms to about 45micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 0.1 to about 500 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 1 to about350 micrograms of nucleic acid molecule. In some embodiments, thecompositions contain about 25 to about 250 micrograms of nucleic acidmolecule. In some embodiments, the compositions contain about 100 toabout 200 micrograms of nucleic acid molecule.

The compositions can be formulated according to the mode ofadministration to be used. In cases where compositions are injectablepharmaceutical compositions, they are sterile, pyrogen free andparticulate free. An isotonic formulation can be used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are suitable. Stabilizers include gelatinand albumin. In some embodiments, a vasoconstriction agent is added tothe formulation.

The compositions can further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient can be functionalmolecules as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient can be a transfection facilitatingagent, which can include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freund's incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalane, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents. The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and more suitably, thepoly-L-glutamate is present in the composition at a concentration lessthan 6 mg/ml. The transfection facilitating agent can also includesurface active agents such as immune-stimulating complexes (ISCOMS),Freunds incomplete adjuvant, LPS analog including monophosphoryl lipidA, muramyl peptides, quinone analogs and vesicles such as squalene andsqualane, and hyaluronic acid can also be used administered inconjunction with the genetic construct. In some embodiments, the plasmidcompositions can also include a transfection facilitating agent such aslipids, liposomes, including lecithin liposomes or other liposomes knownin the art, as a DNA-liposome mixture (see for example W09324640),calcium ions, viral proteins, polyanions, polycations, or nanoparticles,or other known transfection facilitating agents. In some embodiments,the transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. Concentration of thetransfection agent in the composition is less than 4 mg/ml, less than 2mg/ml, less than 1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml,less than 0.250 mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, orless than 0.010 mg/ml.

The pharmaceutically acceptable excipient may be an adjuvant. Theadjuvant may be other genes that are expressed in alternative plasmid orare delivered as proteins in combination with the plasmid above. Theadjuvant may be selected from the group consisting of: α-interferon(IFN-α), β-interferon (IFN-β), γ-interferon, platelet derived growthfactor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF),cutaneous T cell-attracting chemokine (CTACK), epithelialthymus-expressed chemokine (TECK), mucosae-associated epithelialchemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 including IL-15 havingthe signal sequence deleted and optionally including the signal peptidefrom IgE. The adjuvant may be IL-12, IL-15, IL-28, CTACK, TECK, plateletderived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growthfactor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or acombination thereof.

Other genes which may be useful adjuvants include those encoding: MCP-1,MIP-1a, MIP-1p, IL-8, L-selectin, P-selectin, E-selectin, CD34,GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2,ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40,CD40L, vascular growth factor, fibroblast growth factor, IL-7, nervegrowth factor, vascular endothelial growth factor, Fas, TNF receptor,Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5,KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1,Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1,JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof.

The plasmid compositions can further comprise a genetic vaccinefacilitator agent as described in U.S. Ser. No. 021,579 filed Apr. 1,1994, which is fully incorporated by reference.

The present disclosure also provides kits comprising any of the Mtbantigens, fragments thereof, fusion proteins, nucleic acid molecules,CMV vectors, or cells, described herein. The kit can include, forexample, container(s), package(s) or dispenser(s) along with labels andinstructions for administration or use.

Vaccine CMV vectors and pharmaceutical compositions may be presented inunit-dose or multi-dose containers, such as sealed ampoules or vials.Such containers can be hermetically sealed to preserve sterility of theformulation until use. In general, formulations may be stored assuspensions, solutions or emulsions in oily or aqueous vehicles.Alternatively, a vaccine or pharmaceutical composition may be stored ina freeze-dried condition requiring only the addition of a sterile liquidcarrier immediately prior to use.

The present disclosure also provides methods for treatment or preventionof tuberculosis comprising administering to a subject in need thereof atleast one recombinant RhCMV or HCMV vaccine vector as described herein.In some embodiments, the methods further comprise re-administering tothe subject at least one recombinant RhCMV or HCMV vaccine vectordescribed herein. In some embodiments, the recombinant RhCMV or HCMVvaccine vector of the re-administration is different than therecombinant RhCMV or HCMV vaccine vector of the initial administration.

The present disclosure also provides methods for eliciting an immuneresponse to a Mtb antigen comprising administering to a subject in needthereof at least one recombinant RhCMV or HCMV vaccine vector asdescribed herein.

The present disclosure also provides methods for eliciting a CD8⁺ orCD4⁺ T cell response to a Mtb antigen comprising administering to asubject in need thereof at least one recombinant RhCMV or HCMV vaccinevector as described herein.

In some embodiments, the recombinant RhCMV or HCMV vaccine vector isadministered to the subject intravenously, intramuscularly,intraperitoneally, intranasally, or orally. In some embodiments, thesubject is a human.

In some embodiments, any of the Mtb antigens, constructs, vectors, orcells described herein, or compositions comprising the same, can beadministered to a mammal as an aerosol. In some embodiments, the aerosolinocula comprises saline. Conventional aerosol delivery devices include,but are not limited to, a pressurized metered dose inhaler (pMDI) and adry power inhaler (DPI), both of which deliver a dry powder formulation,and nebulizers such as the PARI eFlow device, which delivers an aqueousdose as a fine mist. In some embodiments, the aerosol delivery device isa Pari eFlow portable electronic aerosol delivery platform attached to adelivery mask. In some embodiments, the average particle size is fromabout 1 μm to about 10 μm, from about 1 μm to about 5 μm, from about 3μm to about 5 μm, from about 4 μm to about 5 μm, or from about 3.9 μm toabout 4.9 μm. In some embodiments, the aerosol is in a volume from about0.1 ml to about 5 ml, from about 0.1 ml to about 2 ml, from about 0.1 mlto about 1.5 ml, from about 0.5 ml to about 1.5 ml, from about 0.5 ml toabout 1.2 ml, from about 0.7 ml to about 1.2 ml, or about 1 ml.

Effective doses of the compositions of the present disclosure, for thetreatment of a condition vary depending upon many different factors,including means of administration, target site, physiological state ofthe subject, whether the subject is human or an animal, othermedications administered, and whether treatment is prophylactic ortherapeutic. Usually, the subject is a human but non-human mammalsincluding transgenic mammals can also be treated.

In some embodiments, the compositions can be administered to a subjectby injection intravenously, subcutaneously, intraperitoneally,intramuscularly, intramedullarily, intraventricularly, intraepidurally,intraarterially, intravascularly, intraarticularly, intrasynovially,intrasternally, intrathecally, intrahepatically, intraspinally,intratumorly, intracranially, enteral, intrapulmonary, transmucosal,intrauterine, sublingual, or locally at sites of inflammation or tumorgrowth by using standard methods. Alternately, the compositions can beadministered to a subject by routes including oral, nasal, ophthalmic,rectal, or topical. The most typical route of administration isintravascular, subcutaneous, or intramuscular, although other routes canbe effective. In some embodiments, compositions are administered as asustained release composition or device, such as a Medipad™ device. Thecomposition can also be administered via the respiratory tract, forexample, using a dry powder inhalation device, nebulizer, or a metereddose inhaler. The composition can also be administered by traditionalsyringes, needleless injection devices, “microprojectile bombardmentgone guns,” or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound.

In some embodiments, the pharmaceutical compositions may be delivered byintranasal sprays, inhalation, and/or other aerosol delivery vehicles.Methods for delivering genes, nucleic acids, and peptide compositionsdirectly to the lungs via nasal aerosol sprays has been described e.g.,in U.S. Pat. Nos. 5,756,353 and 5,804,212 (each specificallyincorporated herein by reference in its entirety). Likewise, thedelivery of drugs using intranasal microparticle resins (Takenaga etal., 1998) and lysophosphatidyl-glycerol compounds (U.S. Pat. No.5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroethylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

In some embodiments, the composition can be administered to a subject bysustained release administration, by such means as depot injections oferodible implants directly applied during surgery or by implantation ofan infusion pump or a biocompatible sustained release implant into thesubject. Alternately, the composition can be administered to a subjectby injectable depot routes of administration, such as by using 1-, 3-,or 6-month depot injectable or biodegradable materials and methods, orby applying to the skin of the subject a transdermal patch containingthe composition, and leaving the patch in contact with the subject'sskin, generally for 1 to 5 hours per patch.

The present disclosure also provides methods of eliciting an immuneresponse against Mycobacterium tuberculosis in a mammal comprisingadministering to the mammal an immunologically sufficient amount of oneor more CMV vectors comprising one or more of the Mtb fusion proteinsdescribed herein.

The fusion proteins and compositions described herein can be used totreat or prevent tuberculosis. In some embodiments, the method comprisesadministering to a human a therapeutically- orprophylactically-effective amount of any of the CMV vectors orcompositions described herein such that the tuberculosis infection isdiminished or prevented.

In some embodiments, the subject being treated will have been previouslydiagnosed as having tuberculosis. Such subjects will, thus, have beendiagnosed as being in need of such treatment. Alternately, the treatmentmay be intended to prevent a tuberculosis infection in a subject thatdoes not yet have tuberculosis or to a subject that is travelling to anarea where tuberculosis is prevalent.

Treatment of a subject suffering from tuberculosis can be monitoredusing standard methods. Some methods entail determining a baselinevalue, for example, of an antibody level or profile in a subject, beforeadministering a dosage of agent, and comparing this with a value for theprofile or level after treatment. A significant increase such as, forexample, greater than the typical margin of experimental error in repeatmeasurements of the same sample, expressed as one standard deviationfrom the mean of such measurements in value of the level or profilesignals a positive treatment outcome (i.e., that administration of theagent has achieved a desired response). If the value for immune responsedoes not change significantly, or decreases, a negative treatmentoutcome is indicated.

In other embodiments, a control value such as a mean and standarddeviation, of level or profile is determined for a control population.Typically the individuals in the control population have not receivedprior treatment. Measured values of the level or profile in a subjectafter administering a therapeutic agent are then compared with thecontrol value. A significant increase relative to the control value,such as greater than one standard deviation from the mean, signals apositive or sufficient treatment outcome. A lack of significant increaseor a decrease signals a negative or insufficient treatment outcome.Administration of the therapeutic is generally continued while the levelis increasing relative to the control value. As before, attainment of aplateau relative to control values is an indicator that theadministration of treatment can be discontinued or reduced in dosageand/or frequency.

In other embodiments, a control value of the level or profile, such as amean and standard deviation, is determined from a control population ofindividuals who have undergone treatment with a therapeutic agent andwhose levels or profiles have plateaued in response to treatment.Measured values of levels or profiles in a subject are compared with thecontrol value. If the measured level in a subject is not significantlydifferent, such as by more than one standard deviation, from the controlvalue, treatment can be discontinued. If the level in a subject issignificantly below the control value, continued administration of agentis warranted. If the level in the subject persists below the controlvalue, then a change in treatment may be indicated.

In other embodiments, a subject who is not presently receiving treatmentbut has undergone a previous course of treatment is monitored forantibody levels or profiles to determine whether a resumption oftreatment is required. The measured level or profile in the subject canbe compared with a value previously achieved in the subject after aprevious course of treatment. A significant decrease relative to theprevious measurement, such as greater than a typical margin of error inrepeat measurements of the same sample, is an indication that treatmentcan be resumed. Alternately, the value measured in a subject can becompared with a control value (mean plus standard deviation) determinedin a population of subjects after undergoing a course of treatment.Alternately, the measured value in a subject can be compared with acontrol value in populations of prophylactically treated subjects whoremain free of symptoms of disease, or populations of therapeuticallytreated subjects who show amelioration of disease characteristics. Inall of these cases, a significant decrease relative to the controllevel, such as more than a standard deviation, is an indicator thattreatment should be resumed in a subject.

In some methods, a baseline measurement of antibody to a given antigenin the subject is made before administration, a second measurement ismade soon thereafter to determine the peak antibody level, and one ormore further measurements are made at intervals to monitor decay ofantibody levels. When the level of antibody has declined to baseline ora predetermined percentage of the peak less baseline, such as 50%, 25%or 10%, administration of a further dosage of antigen is administered.In some embodiments, peak or subsequent measured levels less backgroundare compared with reference levels previously determined to constitute abeneficial prophylactic or therapeutic treatment regime in othersubjects. If the measured antibody level is significantly less than areference level, such as less than the mean minus one standard deviationof the reference value in population of subjects benefiting fromtreatment, administration of an additional dosage of antigen isindicated.

Immunization schedules (or regimens) are well known for animals(including humans) and can be readily determined for the particularsubject and immunogenic composition. Hence, the immunogens can beadministered one or more times to the subject. Preferably, there is aset time interval between separate administrations of the immunogeniccomposition. While this interval varies for every subject, typically itranges from 10 days to several weeks, and is often 2, 4, 6 or 8 weeks.For humans, the interval is typically from 2 to 6 weeks. In aparticularly advantageous embodiment of the present invention, theinterval is longer, advantageously about 10 weeks, 12 weeks, 14 weeks,16 weeks, 18 weeks, 20 weeks, 22 weeks, 24 weeks, 26 weeks, 28 weeks, 30weeks, 32 weeks, 34 weeks, 36 weeks, 38 weeks, 40 weeks, 42 weeks, 44weeks, 46 weeks, 48 weeks, 50 weeks, 52 weeks, 54 weeks, 56 weeks, 58weeks, 60 weeks, 62 weeks, 64 weeks, 66 weeks, 68 weeks or 70 weeks.

In some embodiments, the subject(s) that can be treated by theabove-described methods is an animal, such as a mammal, including, butare not limited to, humans, non-human primates, rodents (including rats,mice, hamsters and guinea pigs) cow, horse, sheep, goat, pig, dog andcat. In most instances, the mammal is a human.

The present disclosure also provides CMV/TB vectors as described hereinfor use in the preparation of a medicament for treating or preventing aMycobacterium tuberculosis infection.

The present disclosure also provides CMV/TB vectors as described hereinfor use in treating or preventing a Mycobacterium tuberculosisinfection.

The present disclosure also provides uses of CMV/TB vectors as describedherein in the preparation of a medicament for treating or preventing aMycobacterium tuberculosis infection.

The present disclosure also provides uses of CMV/TB vectors as describedherein in treating or preventing a Mycobacterium tuberculosis infection.

The present disclosure also provides any of the CMV/TB vectors asdescribed herein, or any of the compositions described herein, or any ofthe cells described herein, or any of the methods described herein, orany of the uses described herein, substantially as described withreference to the accompanying examples and/or figures.

The following representative embodiments are presented:

Embodiment 1

A recombinant RhCMV or HCMV vector comprising a nucleic acid sequenceencoding an expressible Mtb antigen selected from Ag85A-Ag85B-Rv3407,Rv1733-Rv2626c, RpfA-RpfC-RpfD, Ag85B-ESAT6, andAg85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.

Embodiment 2

The recombinant RhCMV or HCMV vaccine vector of embodiment 1, whereinexpression of the Mtb antigen is driven by an antigen-coding sequence inoperable association with a promoter selected from the group consistingof a constitutive CMV promoter, an immediate early CMV promoter, anearly CMV promoter, and a late CMV promoter.

Embodiment 3

The recombinant RhCMV or HCMV vaccine vector of embodiment 2, whereinthe promoter is selected from the group consisting of EF1-alpha, UL82,MIE, pp65, and gH.

Embodiment 4

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 3, comprising a deletion or modification of US2, US3, US4, US5, US6,US11, or UL97, or a homolog thereof.

Embodiment 5

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 4, comprising a deletion of Rh158-166 or a homolog thereof.

Embodiment 6

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 5, wherein the RhCMV or HCMV vaccine vector is a tropism-restrictedvector.

Embodiment 7

The recombinant RhCMV or HCMV vaccine vector of embodiment 6, whereinthe tropism-restrictive vector lacks genes required for optimal growthin certain cell types or contains targets for tissue-specific micro-RNAsin genes essential for viral replication or wherein thetropism-restrictive vector has an epithelial, central nervous system(CNS), or macrophage deficient tropism, or a combination thereof.

Embodiment 8

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 7, wherein the RhCMV or HCMV vaccine vector has a deletion in a generegion non-essential for growth in vivo.

Embodiment 9

The recombinant RhCMV or HCMV vaccine vector of embodiment 8, whereinthe gene region is selected from the group consisting of the RL11family, the pp65 family, the US12 family, and the US28 family.

Embodiment 10

The recombinant RhCMV vaccine vector of embodiment 9, wherein the RhCMVgene region is selected from the group consisting of Rh13-Rh29,Rh111-Rh112, Rh191-Rh202, and Rh214-Rh220, or wherein the RhCMV generegion is selected from the group consisting of Rh13.1, Rh19, Rh20,Rh23, Rh24, Rh112, Rh190, Rh192, Rh196, Rh198, Rh199, Rh200, Rh201,Rh202, and Rh220.

Embodiment 11

The recombinant HCMV vaccine vector of embodiment 9, wherein the HCMVgene region is selected from the group consisting of RL11, UL6, UL7,UL9, UL11, UL83 (pp65), US12, US13, US14, US17, US18, US19, US20, US21,and UL28.

Embodiment 12

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 11, wherein the vector comprises a deletion in a RhCMV or HCMV genethat is essential for replication within a host, dissemination within ahost, or spreading from host to host.

Embodiment 13

The recombinant RhCMV or HCMV vaccine vector of embodiment 12, whereinthe essential gene is UL94, UL32, UL99, UL115, or UL44, or a homologthereof.

Embodiment 14

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 13, wherein the vector comprises a deletion in gene UL82/pp71 or ahomolog thereof.

Embodiment 15

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 14, wherein the vector further comprises a second nucleic acidsequence encoding US2, US3, or US6, or a homolog thereof, wherein thevector does not encode a functional US11.

Embodiment 16

The recombinant RhCMV or HCMV vaccine vector of embodiment 15, whereinthe second nucleic acid sequence encodes US2, US3, and US6.

Embodiment 17

The recombinant RhCMV or HCMV vaccine vector of embodiment 15 orembodiment 16, wherein the nucleic acid encoding a US11 open readingframe is deleted.

Embodiment 18

The recombinant RhCMV or HCMV vaccine vector of any one of embodiment s15 to 17, further comprising a third nucleic acid sequence encodingUS11, and wherein the nucleic acid sequence encoding US11 comprises apoint mutation, a frameshift mutation, and/or a deletion of one or morenucleotides of the nucleic acid sequence encoding US11.

Embodiment 19

The recombinant RhCMV or HCMV vaccine vector of embodiment 18, whereinthe vector lacks the tegument protein pp65.

Embodiment 20

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 19, wherein the vector does not express an active UL130 protein.

Embodiment 21

The recombinant RhCMV or HCMV vaccine vector of any one of embodiments 1to 20, wherein the RhCMV vaccine vector is Rh68-1 or Rh68-1.2.

Embodiment 22

The recombinant RhCMV or HCMV vaccine vector of embodiment 1 furthercomprising a microRNA recognition element (MRE) operably linked to a CMVgene that is essential or augmenting for CMV growth, and wherein the MREsilences expression in the presence of a microRNA that is expressed by acell of myeloid lineage.

Embodiment 23

A pharmaceutical composition comprising the recombinant RhCMV or HCMVvaccine vector of any one of embodiments 1 to 22, and a pharmaceuticallyacceptable carrier.

Embodiment 24

A method for treatment or prevention of tuberculosis comprisingadministering to a subject in need thereof at least one recombinantRhCMV or HCMV vaccine vector of any one of embodiments 1 to 22.

Embodiment 25

The method of embodiment 24, further comprising re-administering to thesubject at least one recombinant RhCMV or HCMV vaccine vector of any oneof embodiments 1 to 22.

Embodiment 26

The method of embodiment 25, wherein the recombinant RhCMV or HCMVvaccine vector of the re-administration is different than therecombinant RhCMV or HCMV vaccine vector of the initial administration.

Embodiment 27

A method for eliciting an immune response to a Mtb antigen comprisingadministering to a subject in need thereof at least one recombinantRhCMV or

HCMV vaccine vector of any one of embodiments 1 to 22.

Embodiment 28

A method for eliciting a CD8+ or CD4+ T cell response to a Mtb antigencomprising administering to a subject in need thereof at least onerecombinant RhCMV or HCMV vaccine vector of embodiment 20.

Embodiment 29

The method of any one of embodiments 24 to 28 wherein the recombinantRhCMV or HCMV vaccine vector is administered to the subjectintravenously, intramuscularly, intraperitoneally, intranasally, ororally.

Embodiment 30

The method of any one of embodiments 24 to 29 wherein the vector is anHCMV vector and the subject is a human.

Embodiment 31

A Mtb antigen selected from Ag85B-ESAT6 andAg85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.

Embodiment 32

The Mtb antigen of embodiment 31 which isAg85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.

In order that the subject matter disclosed herein may be moreefficiently understood, examples are provided below. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the claimed subject matter in anymanner Throughout these examples, molecular cloning reactions, and otherstandard recombinant DNA techniques, were carried out according tomethods described in Maniatis et al., Molecular Cloning—A LaboratoryManual, 2nd ed., Cold Spring Harbor Press (1989), using commerciallyavailable reagents, except where otherwise noted.

EXAMPLES Example 1: CMV/TB Vectors

Particular aspects provide recombinant HCMV/TB vectors that can begrowth-modulated in vivo (e.g., by oral administration of the antibioticdoxycycline). Heterologous antigen expression may be under the controlof promoters of different kinetic classes with respect to the CMVinfection cycle (e.g., EF1α—constitutive; MIE—immediate early;pp65—early; gH—late).

In particular embodiments, HCMV/TB vectors lack immune modulatory genes(e.g., Rh158-166 and Rh182-189) to enhance vector immunogenicity, safetyand heterologous gene carrying capacity of the vector. For example, HCMVencodes at least four different gene products, gpUS2, gpUS3, gpUS6 andgpUS11 that interfere with antigen presentation by MHC I. All four HCMVMHC evasion molecules are encoded in the unique short region of HCMV andbelong to the related US6 gene family Additional HCMV immunomodulatorsinclude, but are not limited to UL118, UL119, UL36, UL:37, UL111a,UL146, UL147, etc. Likewise, RhCMV contains analogous immune modulatorygenes, that can be deleted or modified to enhance vector immunogenicity,safety and heterologous gene carrying capacity of the inventive vaccinevectors.

In additional embodiments, HCMV/TB are further optimized for anti-TBimmunogenicity by insertion of multiple antigen genes, such as thosedisclosed herein. Alternatively, several vectors, each having a singleinserted antigen may be used for co-administration.

In additional embodiments, HCMV/TB vectors contain LoxP sitesstrategically placed in the CMV genome to flank an essential region ofthe viral genome, in combination with a tetracycline (Tet)-regulated Crerecombinase.

Construction and Characterization of the RhCMV BAC.

The development of BAC technology to clone large segments of genomic DNAcoupled with sophisticated λ phage-based mutagenesis systems hasrevolutionized the field of herpes virology enabling genetic approachesto analyze the virus. Applicants have used this system, for example, toconstruct an RhCMV BAC(RhCMV BAC-Cre) containing the complete RhCMVstrain 68-1 genome. The RhCMV BAC-Cre was derived from an infectious,pathogenic RhCMV 68-1/EGFP recombinant virus. RhCMV BAC-Cre contains aBAC cassette inserted at a single LoxP site within the Rh181 (US1)/Rh182(US2) intergenic region of RhCMVvLoxP. Insertion of the BAC cassette atthis site results in the generation of LoxP sequences flanking thecassette. As the BAC cassette contains a Cre gene that is expressed ineukaryotic cells, transfection of this “self-excising” RhCMV BAC-Creinto fibroblasts results in efficient excision of the BAC cassette,reconstituting virus (designated RhCMVvLoxP). Characterization of thegrowth of the BAC-reconstituted virus (RhCMVvLoxP) in vitro and in vivodemonstrates that the various genetic manipulations did not alter the WTproperties of the virus. The genomic structure of RhCMVvLoxP isidentical to that of WT RhCMV except for the residual LoxP site. Thepresence of the LoxP sequence does not alter the expression profiles ofneighboring Rh181 (US1) and Rh182 (US2) or distal (IE2) genes.RhCMVvLoxP replicates with WT kinetics both in tissue culture and inRhCMV seronegative immunocompetent RMs (n=2). Analysis of tissues fromone animal terminated at 6 months post-inoculation demonstrated thepresence of both RhCMV DNA and IE1-expressing cells in the spleen,consistent with the persistent gene expression observed in previousstudies with WT virus. Both RMs developed vigorous anti-RhCMV antibodytiters comparable to those observed in naturally infected animals. Takentogether, these observations demonstrate that RhCMVvLoxP isphenotypically WT and is suitable to construct site-specific alterationsfor the development of vaccine vectors.

Example 2: Non-Human Primate Study #1

This challenge included four treatment groups: 1) naive non-humanprimates (NHPs) (e.g., Rhesus Macaques) (i.e., unvaccinated controls)(n=8); 2) NHPs vaccinated with BCG alone (n=7); 3) NHPs vaccinated witha cocktail of CMV/TB vectors alone (n=7); and 4) NHPs vaccinated withBCG and a cocktail of CMV/TB vectors. The naive NHPs wereCMV-seropositive. The NHPs vaccinated with BCG were vaccinated with 0.1ml intradermally with Statens Serum Institute (SSI) BCG vaccine. TheNHPs vaccinated with a cocktail of CMV/TB vectors were vaccinated withfour Rh68-1 vectors (encoding fusion proteins comprised ofAg85A-Ag85B-Rv3407, Rv1733-Rv2626, RpfA-RpfC-RpfD, and Ag85B-ESAT6). TheNHPs vaccinated with BCG and a cocktail of CMV/TB vectors were primedwith the SSI BCG and boosted with the same cocktail of the four Rh68-1vectors as above. The NHPs who received the CMV/TB vectors received5×10⁶ PFU (plaque forming unit) of CMV/TB vector cocktailssubcutaneously at weeks 6 and 21. The two BCG groups were given BCG atWeek 0. At Weeks 6 and 21, the two CMV/TB vectors groups were immunizedwith the cocktail of RhCMV/TB vectors. NHP were challenged withMycobacterium tuberculosis at Week 49. Endpoints included longitudinalCT scanning, gross pathology and bacterial burden.

Three of the four vectors used in this study encode classical, latencyand resuscitation antigen cassettes (encoding fusion proteins comprisedof Ag85A-Ag85B-Rv3407, Rv1733-Rv2626, and RpfA-RpfC-RpfD, respectively).NHP received 5E6 pfu/RhCMV vector, delivered subcutaneously. Alsoincluded was a Rh68-1 vector encoding a fusion protein comprised ofAg85AB and ESAT6.

Immunogenicity of these vectors was evaluated using intracellularcytokine staining FIG. 1 shows CD4+ and CD8+ T cell responses. PBMCswere assayed for each of the 9 antigens.

FIGS. 2A, 2B, 2C, and 2D show ESAT-6-specific responses followingvaccination. FIGS. 3A, 3B, 3C, and 3D show Rv1733-specific responsesfollowing vaccination. FIGS. 4A, 4B, 4C, and 4D show RpfC-specificresponses following vaccination. FIGS. 5A, 5B, 5C, and 5D showAg85B-specific responses following vaccination. FIGS. 6A and 6B showAg85A-specific responses following vaccination. FIGS. 7A and 7B showRv3407-specific responses following vaccination. FIGS. 8A and 8B showRv2626-specific responses following vaccination. FIGS. 9A and 9B showRpm-specific responses following vaccination. FIGS. 10A and 10B showRpfA-specific responses following vaccination.

FIG. 11 shows the phenotypic differentiation of BCG- and RhCMV-induced Tcells in blood.

FIG. 12 shows that these Rh68-1 vectors, as expected, induce CD8 T cellsthat are primarily restricted by MHC II.

The RhCMV vectors were highly immunogenic, showing robust responses inboth peripheral blood mononuclear cells (PBMCs) as well asbronchoalveolar lavage (BAL). Vaccination with Rh68-1 also significantlyreduced disease progression as compared to naive controls.

Efficacy was evaluated by several means, including CT scan analysis(volume of lung involvement), necropsy score (size, number anddistribution of gross lesions; lung, lung-draining lymph nodes, andtotal (which includes distant dissemination)), and necropsyMycobacterium tuberculosis cultures (40 lung samples by sterology (30right; 10 left); 9 lymph nodes (6 lung draining and 3 non-mediastinal);and 9 extra-pulmonary tissues (5 liver, 2 kidney, spleen, andpancreas)).

For pulmonary necropsy scoring, individual lobes were scored on 5 mmsections. The following scores were given for granuloma prevalence: novisible lesions=1; 1-3 lesions=2; 4-10 lesions=3; 11-15 lesions=4; 16-20lesions=5; >20 lesions=6; and Miliary <50% of lobe=7. The followingscores were given for largest granuloma size: none visible=1; <1-2 mm=2;3-4 mm=3; 5-10 mm=4; 11-20 mm=5; >20 mm=6; confluent or miliary lesionsinvolving <50% of lobe=7; and confluent or miliary lesionsinvolving >50% of lobe=8. The following scores were given for additionalscoring criteria (1=absent; 2=present): parietal pleural adhesionsassociated with granulomatous disease, parietal pleural thickeningassociated with granulomatous disease; granulomatous disease withcavitation, and granulomatous disease involving parietal pleura,diaphragm or body wall.

For thoracic lymph node necropsy scoring, the following scores weregiven for size: nodes visible but not enlarged (≤5 mm)=0; nodes visiblyenlarged (≤5-10 mm) (unilateral)=1; nodes visibly enlarged (≤5-10 mm)(bilateral)=2; and nodes visibly enlarged (>1 cm)(unilateral/bilateral)=3. The following scores were given for granulomaprevalence: no granulomas visible on capsular or cut surface=0; focal ormultifocal, circumscribed, non-coalescing granulomas <2 mm=1; coalescingsolid or caseous granulomas occupying <50% of node=2; coalescing solidor caseous granulomas occupying >50% of node=3; and completegranulomatous nodal effacement=4. The following scores were given foradditional scoring criteria (absent=1; present=2): other thoracic lymphnodes.

For liver and spleen necropsy scoring, the following scores were givenfor prevalence: no visible granulomas=0; 1-3 visible granulomas=1; 4-10visible granulomas=2; >10 visible granulomas=3; and miliary pattern=4.The following scores were given for granuloma size: none present=0; <1-2mm=1; 3-4 mm=2; and >4 mm=3.

For miscellaneous organs and tissue, the following scores were given forprevalence: no visible granulomas=0; 1-3 visible granulomas=1; 4-10visible granulomas=2; >10 visible granulomas=3; and miliary pattern=4.The following scores were given for granuloma size: none present=0; <1-2mm=1; 3-4 mm=2; and >4 mm=3.

FIG. 13 shows the correlation of various efficacy criteria (i.e.,necropsy <16 weeks for cause, and >16 weeks randomized) for a bi-weeklyand pre-necropsy CT Scan (lung disease only). The necropsy scoreaccounted for the size, number and distribution of gross lesions in thelung, lung draining lymph nodes, and distal sites. In particular, 40lung samples (30 right; 10 left), 9 lymph nodes (6 lung draining and 3non-mediastinal), and 9 extra-pulmonary tissue (5 liver, 2 kidney,spleen, and pancreas) were analyzed by sterology. FIG. 14 shows CT Scansat 14 weeks post-infection. FIG. 15 shows the quantification ofpulmonary disease by CT scan. FIG. 16 shows the gross pathology scores.These scores reflect the number of granulomas present, the size of thegranulomas, and any complex pathologies (pleural thickening, pneumonia,etc). Included are overall scores (which include lung, lung draininglymph nodes, liver and spleen), as well as separated scores for the lungand lung draining lymph nodes. FIGS. 17 and 18 show the extent ofdisease and dissemination. RhCMV/TB vector vaccination alone achieved73% efficacy against overall disease spread, which was statisticallysuperior to both unvaccinated and BCG vaccination alone. Monkeys primedwith BCG 6 weeks before RhCMV/TB vaccination were still significantlyprotected against overall disease spread, but protection was less thanthat observed in the RhCMV/TB vaccination alone. FIG. 19 shows theextent of disease and dissemination in non-lung samples. FIG. 20 showsthe extent of disease and dissemination in lymph nodes.

FIG. 21 shows comparative T cell response analysis. FIG. 22 showscorrelation of CD4 T cell responses with extrapulmonary spread. FIG. 23shows correlation of CD8 T cell responses with extrapulmonary spread. Insummary, the strongest correlate is between peak ESAT6-specific CD4+ Tcell responses in blood after first CMV vector vaccination (correlationweakens but is still significant after exclusion of BCG group). Bloodresponse correlates are strongest for extent of extrapulmonary disease(vs. extent of lung disease). The “best” blood CD4+ responses are:ESAT6, RpfA, RpfD, Rv2626, Rv3407. The “best” blood CD8+ responses: sameas CD4+Ag 85A and 85B. Few BAL responses correlate with outcome.

FIG. 24 shows confirmation of infection by immune analysis, wherebyCFP10, an Mtb antigen not included in the RhCMV constructs, was used toconfirm infection by detection of de novo immune responses. FIG. 25shows post-challenge immune responses in PBMCs.

FIGS. 26 and 27 show CD4 T cell responses post-necropsy. FIG. 28 showsantigen-specific CD4 T cell responses in lymph nodes analyzedpost-necropsy. FIGS. 29 and 30 show CD8 T cell responses post-necropsy.FIG. 31 shows antigen-specific CD8 T cell responses in lymph nodesanalyzed post-necropsy. Overall, TB infection-elicited CFP10-specificCD4+ and CD8+ T cell responses are similar between all RM groups. CMVvectors maintain higher frequencies of TB insert-specific CD4+ T cellsresponses in PBMC, BM, spleen and lung for all inserts except ESAT6.This difference is less apparent in LN samples, except for RpfA and RpfD(although in certain LNs, RpfC, 85A, Rv2623, and Rv3407 Ag responses arealso highest in the CMV vector vaccinated group). TB insert specificCD8+ T cell responses are more variable (and tend to be higher inunvaccinated RM), but responses to RpfA, RpfD, 85A, Rv2626 and Rv3407still tend to be higher in CMV vector-vaccinated animals in mosttissues.

FIG. 32 shows post-necropsy correlation between splenic CD4 T cellresponses and lymph node culture. A correlation of CD4+ responses tonon-lung disease was observed for Ag85A, RpfA, RpfD, Rv2626, and Rv3407(moderate to strong), as well as for Ag85B and RpfC (although weaker). Acorrelation of CD8+ responses to lung disease was observed for RpfA andRpfD (moderate), as well as for ESAT6 (although weaker). A correlationof CD8+ responses to non-lung disease was observed for Ag85A, RpfA, andRpfD (strong), as well as for Rv2626, Rv3407, and Ag85B (moderate). PeakESAT6-specific responses (particularly CD4+) during vaccinationcorrelated with reduced extra-pulmonary disease after challenge, but notat necropsy (as TB infection eventually induced high frequencyESAT6-specific responses in all animals).

In general, CMV vectors elicit and maintain higher TB insert-specificCD4+ and CD8+ T cell responses than BCG. These responses are associatedwith significant protection against both pulmonary and extra-pulmonarydisease progression. BCG was associated with, at best, a trend towardsmodest pulmonary protection, but extra-pulmonary disease was nodifferent than that of unvaccinated controls. Indeed, the administrationof BCG prior to CMV vector vaccination partially abrogated CMVvector-mediated protection, in particular for extra-pulmonary spread. Tcell responses to some, but not all, TB inserts were lower in sometissues in the animals receiving both BCG and CMV vectors compared tothose receiving CMV vectors alone, but it remains to be determinedwhether this or another mechanism (e.g., BCG elicited responsespromoting bacterial spread) account for the reduced protectionassociated the BCG “prime.” Correlates of protection were stronger forextra-pulmonary disease than pulmonary disease and point to RpfA, RpfD,Ag85A, Rv2626 and Rv3407 (and possibly ESAT6) being the most effectivevaccine inserts.

In summary, RhCMV/TB vector-vaccinated RM show significantly reduceddisease (both pulmonary and extra-pulmonary) by all criteria. RhCMV/TBvectors elicited and maintained higher (and qualitatively different) TBinsert-specific CD4+ and CD8+ T cell responses than BCG. RhCMV/TB vectorvaccination provided significant protection against both pulmonary andextra-pulmonary disease progression (73% overall). BCG was associatedwith a trend towards modest pulmonary protection compared tounvaccinated controls, but extra-pulmonary disease was no different thanthat of unvaccinated controls. The combination of BCG and RhCMV/TBvectors is less effective than RhCMV/TB vector vaccination alone withthe BCG component reducing both pulmonary and extra-pulmonaryprotection. RhCMV/TB vector-vaccinated RM may manifest a marked enhancedearly response to infection in carinal LNs. The outcome (extra-pulmonaryspread) predominantly correlated with CD4+ T cell responses to Ag85A,Rpf-A/C/D, Rv3407, Rv2626, and ESAT6.

Example 3: Non-Human Primate Study #2

The second study was designed to confirm and extend previous findings ofCMV-induced protection against Mycobacterium tuberculosis in rhesusmacaques. In the original study, significant protection was induced by acocktail of CMV vectors (strain 68-1), encoding a total of 9 antigens.

The second NHP study consisted of four vaccine groups: 1) Strain 68-1RhCMV/TB-9Ag vector set (n=9); 2) Strain 68-1.2 RhCMV/TB-9Ag vector set(n=9); 3) Strain 68-1 RhCMV/TB-6Ag single vector (n=9); and 4)unvaccinated. Group 1) consisted of Macaques vaccinated with a cocktailof four Rh68-1 vectors (encoding fusion proteins comprised ofAg85A-Ag85B-Rv3407, Rv1733-Rv2626, RpfA-RpfC-RpfD, and Ag85B-ESAT6).Group 2) consisted of Macaques vaccinated with a cocktail of fourRh68-1.2 vectors (encoding fusion proteins comprised ofAg85A-Ag85B-Rv3407, Rv1733-Rv2626, RpfA-RpfC-RpfD, and Ag85B-ESAT6).Group 3) consisted of Macaques vaccinated with a single Rh68-1 vector(encoding a fusion protein comprised ofAg85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD).

At weeks 0 and 14, NHP in Groups 1)-3) were vaccinated with the RhCMV/TBvectors described above. NHP were challenged with Mycobacteriumtuberculosis (Erdman E11-10 mTB at 10 CFU given intrabronchially) atweek 55. There was not BAL post-challenge. The outcomes analyzed werethe same as those described above in Example 2.

The 4 Rh68-1 vectors used in the first study were used again here (seeabove for information on construction). Three of the four Rh68-1.2vectors used in this study encode classical, latency and resuscitationantigen cassettes (encoding fusion proteins comprised ofAg85A-Ag85B-Rv3407, Rv1733-Rv2626, and RpfA-RpfC-RpfD, respectively).The Rh68-1 vector encoding a six-antigen fusion protein (comprisingantigens Ag85A, ESAT6, Rv3407, Rv2626, RpfA and RpfD) and Rh68-1.2vector encoding Ag85B and ESAT6 were also used.

Immunogenicity of these vectors was evaluated using intracellularcytokine staining FIG. 33 shows PBMC immune responses followingvaccination with RhCMV vectors; all vaccines showed robustimmunogenicity. FIG. 34 shows comparing immunogenicity induced by eachRhCMV vector; all vaccines showed equivalent immunogenicity to commoninserts. FIG. 35 shows BAL immune responses following vaccination withRhCMV vectors. In addition, for Strain 68-1 RhCMV/TB vectors(UL128/UL130-deleted), all CD8+ T cell responses were MHC-II- andMHC-E-restricted, whereas for Strain 68-1.2 RhCMV/TB vectors(UL128/UL130-intact), all CD8+ T cell responses were MHC-Ia-restricted(data not shown).

FIG. 36 shows Peripheral blood CFP10-specific T cell responses post-Mtbchallenge. All monkeys showed de novo responses to Mtb post challenge.FIG. 37 shows clinical outcome data from longitudinal CT scans, which isused to quantify the volume of lung disease present post-challenge.There is clear evidence of protection in each of the vaccinated groups.FIGS. 38 and 39 show a necropsy score and culture, respectively. FIG.shows overall efficacy of Strain 68-1 RhCMV/TB vectors. FIG. 41 showslate disease resolution in a Strain 68-1 RhCMV/TB-6Ag single vectorvaccination. FIG. 42 shows, at necropsy, the monkey manifested apathologic extent of a disease score of 10 with only a single Mtbculture. The data suggests that Strain 68-1 RhCMV vector-elicited T cellresponses may not only be able to prevent development of TB lesions, butmay also be capable of mediating their regression.

In summary, low dose (10 bacteria) Erdman E11-10 strain mTB challengeresulted in considerably less aggressive disease than previous studiesusing a >25 bacteria challenge dose, but all unvaccinated controls stillshowed both pulmonary and draining LN disease. Strain 68-1 RhCMV/TBvector vaccination, either the 9 Ag vector set, or the single 6 Aginsert vector, provided striking protection against both pulmonary andextra-pulmonary disease after low dose mTB challenge: 1) 10 of 18 NHPs(56%) with no pathologic evidence of infection (7 of which were alsocompletely culture negative) vs. 0% of unvaccinated controls; and 2) 12of 18 NHPs (67%) with less disease (as measured by both Path Score andCulture) than the unvaccinated control with the least extent of diseaseprogression; and 3) efficacy of the Strain 68-1 RhCMV/TB-6Ag singlevector ≥Strain 68-1 RhCMV/TB-9Ag vector set. Strain 68-1.2 RhCMV/TB-9Agvaccination resulted in significant overall protection.

In addition, Strain 68-1 RhCMV/TB vector vaccination protects highly TBsusceptible rhesus macaques from progressive pulmonary andextra-pulmonary TB disease after both high and low dose intrabronchialErdman strain mTB challenge. In the high dose challenge model, BCG wasnot significantly protective and the combination of BCG and Strain 68-1RhCMV/TB vector vaccination was less protective than Strain 68-1RhCMV/TB vector vaccination alone. Current data suggest vector-elicitedCD4+ T cell responses are the primary protective correlate. To date, thesingle 6 Ag (polyprotein) expressing RhCMV vector provides the bestoverall protection, but it remains possible that different or additionalTB Ag inserts would increase efficacy.

Example 4: General Methodology for Studies #3 and #4

Rhesus Macaques:

Sixty-five purpose-bred, pedigreed, male RM (Macaca mulatta) of Indiangenetic background were used in studies 3 and 4. At assignment, these RMwere specific-pathogen free (SPF) as defined by being free of Macacineherpesvirus 1, D-type simian retrovirus, simian T-lymphotrophic virustype 1, simian immunodeficiency virus, and Mtb. All RM used in thisstudy were housed at the Oregon National Primate Center (ONPRC) inAnimal Biosafety level (ABSL)-2 (vaccine phase) and ABSL-3 rooms(challenge phase) with autonomously controlled temperature, humidity,and lighting. RM were single cage housed due to the infectious nature ofthe study and had visual, auditory and olfactory contact with otheranimals. Because the RM were single cage housed, an enhanced enrichmentplan was designed and overseen by nonhuman primate behavior specialists.RM were fed commercially prepared primate chow twice daily and receivedsupplemental fresh fruit or vegetables daily. Fresh, potable water wasprovided via automatic water systems. All RM were observed twice dailyto assess appetite, attitude, activity level, hydration status andevidence of disease (tachypnea, dyspnea, coughing). Physical examsincluding body weight and complete blood counts were performed at allprotocol time points. RM care and all experimental protocols andprocedures were approved by the ONPRC. The ONPRC is a Category Ifacility. The Laboratory Animal Care and Use Program at the ONPRC isfully accredited by the American Association for Accreditation ofLaboratory Animal Care (AAALAC), and has an approved Assurance(#A3304-01) for the care and use of animals on file with the NIH Officefor Protection from Research Risks. The IACUC adheres to nationalguidelines established in the Animal Welfare Act (7 U.S.C. Sections2131-2159) and the Guide for the Care and Use of Laboratory Animals (8thEdition) as mandated by the U.S. Public Health Service Policy.

Animal Procedures:

RM were sedated with ketamine HCl or Telazol® for intradermal andsubcutaneous vaccine administration, venipuncture, bronchoalveolarlavage, lymph node biopsy, intrabronchial Mtb inoculation and computedtomography (CT) procedures. Mtb Erdman K01 was diluted in saline,lightly sonicated and bacteria were delivered to a segmental bronchus inthe right caudal lung lobe using a bronchoscope. The RM in Studies 3 and4 received 25 and 10 colony forming units (CFU), respectively, in avolume of 2 ml. Pre- and post-challenge axial CT scans (2.5 mm slices)were obtained using a multi-section CT scanner using helical technique,collimation 3 mm and pitch 1.5 (CereTom, Neurologica Corp., Danvers,Mass.) and reconstructed as 1.25 mm slices to improve detectionsensitivity. Nonionic iodinated contrast (Isovue 370, 1-2 ml/kg, BraccoDiagnostics, Princeton N.J.) was administered IV at a rate of 1-2 ml/s.CT scans were obtained with 120 kVp and 200 mA. All animals were imagedpre-challenge, at two-week intervals for the duration of the studies andimmediately prior to necropsy. Scans were interpreted by a veterinarianwho was blinded to the identity of the subject. Lesion area insequential scans was determined from transverse slices through entirelung fields using the IMPAX 6.5.5.3020 software area tool (AGFAHealthCare N.V., Mortsel, Belgium) and lesion volume determined bymultiplying area by 1.25. One RM developed extensive bilateral miliarydisease with estimated lesion volume >200,000 mm³ and no furtherattempts to estimate lesion volume were made in this animal.

Necropsy:

The humane criteria for removing RM with end-stage TB from the studiesare as follows: 1) marked lethargy, 2) severe dyspnea at rest and/orfailure to maintain adequate oxygenation (85%) based on pulse oximetryor blood gas analysis, 3) hemoptysis, 4) weight loss (>15% in 2weeks; >25% over any time course in an adult animal), 5) hypothermia<96° F. with supplemental heating, 6) persistent anemia (<20% for 2weeks), 7) dehydration unresponsive to oral rehydration therapy for 3days, 8) non-responsiveness to therapy for spontaneous diseasesconditions, 9) poor appetite, requiring more than 3 orogastric tubefeedings in 7 days, 10) obtundation, 11) neurologic deficits, and 12)persistent self-injurious behavior unresponsive to a change in locationor enrichment. RM that manifested one or more of these end-stagecriteria were immediately necropsied. RM that remained clinically wellafter post-infection week 16 were randomized and scheduled foreuthanasia and necropsy at the rate of two per week. There were 3exceptions to this general rule of necropsy initiated by end-stagedisease criteria or randomization (designated as “other”). Onenon-end-stage RM (D1) in Study 3 was euthanized on the same day as anend-stage RM because the IACUC does not permit housing a single RM alonein a room. Two additional RM (N1, N3) in Study 3 were euthanized becauseof failure to maintain adequate oxygenation following a bronchoalveolarlavage procedure that was not attributable to end-stage Mtb disease. Toavoid this issue in Study 4, BALs were not performed after challenge inthis study.

At the humane or scheduled endpoint RM were euthanized with sodiumpentobarbital overdose (>50 mg/kg) and exsanguinated via the distalaorta. The necropsy procedure included complete gross pathologicevaluation of abdominal organs and tissues and the brain prior toentering the thoracic cavity to avoid contamination. Macroscopicgranulomas in liver, spleen, kidney and the brain were counted, measuredand photographed in serial 5 mm tissue slices whereas granulomasoccurring in extra thoracic lymph nodes, the gastrointestinal tract andsoft tissues were collected, measured and photographed with minimumsectioning and given a numeric point value score using asemi-quantitative grading system (see, FIG. 48). Granulomas (≤10)occurring in these tissues were bisected and one half collected inpre-weighed sterile media tubes for mycobacterial culture (see below)and the remaining half immersed in 10% neutral-buffered formalin forhistologic analysis. Representative granulomas were selected formycobacterial culture and histology from tissues with >10 granulomas.Single small granulomas (≤1 mm) were utilized entirely for bacteriology.Representative samples of all abdominal organs and tissues and the brainwere collected and then fixed in 10% neutral-buffered formalin forhistologic analysis. Additional samples from these tissues werecollected for mononuclear isolation and for mycobacterial culture. Thepleura and thoracic wall were examined on entering the thoracic cavityand macroscopic granulomas and adhesions were collected, counted,measured, photographed, scored as described (see, FIG. 48) and sampledfor quantitative bacteriology and histology. The thoracic viscera wereremoved en bloc, and then transferred to a sterile cutting board forexamination and dissection. Extreme care was taken to avoidmycobacterial contamination of thoracic tissues by spillage of granulomacontents during dissection, and this was accomplished in all but 1 RM inwhich gross spillage of granuloma contents was observed upon opening thethoracic cavity. The heart was removed and examined and pulmonary andmediastinal lymph nodes and individual lung lobes were dissected free,weighed and photographed. Lymph nodes were divided into samples formycobacterial culture, histopathology, and mononuclear cell isolation(with mycobacterial culture prioritized if tissue was limiting).Macroscopic granulomas in individual lung lobes were counted, measured,photographed and scored in serial 5 mm tissue slices. Samples formycobacterial culture and histology from the right and left lung sliceswere harvested using a nonbiased stereologic sampling method. Individual6 mm lung tissue cores (30 from the right lung and from the left lung)were bisected and one-half collected in pre-weighed sterile media tubesfor bacteriology and the remaining half immersed in 10% neutral-bufferedformalin for histologic analysis. Additionally, representative samplesof all lung lobes and the heart were collected and immersed in 10%neutral-buffered formalin. Tissues for histologic analysis wereroutinely processed and embedded in paraffin. Sections (6 mm) werestained with hematoxylin and eosin. Selected tissues were stained by theZiehl-Neelsen method for acid-fast bacteria.

Vaccines:

The 68-1 and 68-1.2 RhCMV/TB vectors were constructed by bacterialartificial chromosome (BAC) recombineering and were reconstituted andamplified into vector preparations as previously described (Hansen etal., Nature, 2011, 473, 523-527; Hansen et al., Nature, 2013, 502,100-104; Hansen et al., Science, 2013, 340, 1237874; and Hansen et al.,Nat. Med., 2009, 15, 293-299). The Mtb Ags to be included in thesevectors were selected by a bioinformatics selection criteria startingwith the scoring of 4000 Mtb open reading frames by 11 criteria (Zvi etal., BMC Med. Genomics, 2008, 1, 18). Those criteria includedimmunogenicity, vaccine efficacy, expression in granulomas, secretion,and role in hypoxic survival. The top candidates were then furtherscreened by a deeper bioinformatics analysis of predicted T cellepitopes and curated to include antigens that are active duringdifferent stages of TB infection. In addition, all antigens had beenshown to be at least partially protective in a mouse challenge model.The final choice of 9 Mtb proteins for the vaccine inserts included 3representative proteins from so-called acute phase (85A, 85B, ESAT6),latency (Rv1733c, Rv3407, Rv2626c) and resuscitation (Rpf A, Rpf C andRpfD) class of Mtb Ags. These 9 Ags were expressed in 4 differentRhCMV/TB vectors (to be used in combination) for both the 68-1 and68-1.2 backbones, as follows: 1) Ag85A/Ag85B/Rv3407 (GenBank #KY611401),2) Rv1733/Rv26226 (GenBank #KY611402), 3) RpfA/RpfC/RpfD (GenBank#KY611403), and 4) Ag85B/ESAT-6 (GenBank #KY611404). The GenBankAccession #s correspond the to the sequences as they are found in thefinal vectors. Polyprotein #s 1-3 were inserted into the nonessentialRh211 open reading frame under the control of murine CMV IE promotor.Polyprotein #4 was inserted in the same region of the RhCMV genome butunder the control of the EF1α promotor (see, FIG. 46, panel a). For thesingle 6 Ag-expressing, 68-1 RhCMV/TB vector, a single polyproteininsert consisting of 2 Ags from each of 3 classes described above(acute: ESAT-6, Ag85A; latency: Rv3407, Rv2626; resuscitation: RpfA,RpfD; GenBank #KY611405) was used to replace the nonessential Rh107gene, placing its expression under the control of the endogenous Rh107promoter (see, FIG. 46, panels a and b). All BACs were analyzed byrestriction digestion to confirm genomic integrity and were furtherexamined by next generation sequencing (NGS) on an Illumina MiSeqsequencer to ensure the absence of any unintended mutations in thetransgene. To reconstitute the vaccine vectors, the BACs wereelectroporated into telomerized or primary rhesus fibroblasts and keptin culture until full cytopathic effect was achieved. At this point,transgene expression was confirmed by immunoblot of infected celllysates and vaccine stocks were generated by the OHSU Molecular VirologySupport Core (MVSC). Overall genomic integrity and transgene expressionof the final vaccine stocks were confirmed by immunoblots, NGS, andpilot immunogenicity studies in RM. In addition, expression of the ORFsneighboring the TB Ag insertion site was confirmed by RT-PCR. RhCMV/TBvector stocks were titered using primary rhesus fibroblasts in a TCID50assay. Study 3 and 4 RM were vaccinated by subcutaneous administrationof 5×10⁶ pfu of each of the designated RhCMV/TB vectors. For the RMreceiving the 4 vector set, each vector was administered in a separatelimb (right arm, left arm, right leg, left leg). RM were given the testvaccines twice, with the second dose (homologous boost) administered 15weeks (Study 1) or 14 weeks (Study 2) after the first dose. The BCGvaccine (Danish strain 1331; Batch #111005A) was obtained from theStatens Serum Institute (Copenhagen, Denmark) and was reconstituted perthe manufacturer's instructions (Diluent Batch #386587B). RM wereBCG-vaccinated by the intradermal administration of 100 μl of vaccinecontaining 5.5×10⁵ CFUs into the mid-back.

Immunologic Assays:

Mtb-specific CD4+ and CD8+ T cell responses were measured in blood, BALand tissues by flow cytometric ICS, as previously described (Hansen etal., Science, 2013, 340, 1237874; Hansen et al., Science, 2016, 351,714-720; and Hansen et al., Nat. Med., 2009, 15, 293-299). Briefly,mononuclear cell preparations from blood, BAL or tissue were incubatedat 37° C. in a humidified 5% CO₂ atmosphere with overlapping,consecutive 15-mer peptide mixes (11 amino acid overlap) comprisingthese proteins, or individual 15-mer peptides from these proteins, andthe co-stimulatory molecules CD28 and CD49d (BD Biosciences) for 1 hour,followed by addition of brefeldin A (Sigma-Aldrich) for an additional 8hours. Co-stimulation without antigenic peptides served as a backgroundcontrol. As previously described (Hansen et al., Science, 2016, 351,714-720), the MHC restriction (MHC-Ia, MHC-E, MHC-II) of apeptide-specific response was determined by pre-incubating isolatedmononuclear cells for 1 hour at room temperature (prior to addingpeptides and incubating per the standard ICS assay) with the followingblockers: 1) the pan anti-MHC-I mAb W6/32 (10 mg/ml), 2) theMHC-II-blocking CLIP peptide (MHC-II-associated invariant chain, aminoacids 89-100; 20 μM), and 3) the MHC-E-blocking VL9 peptide (VMAPRTLLL;SEQ ID NO:31; 20 μM). Blocking reagents were not washed, but remainthroughout the assay. Following incubation, stimulated cells were fixed,permeabilized and stained as previously described (Hansen et al.,Science, 2013, 340, 1237874; Hansen et al., Science, 2016, 351, 714-720;and Hansen et al., Nat. Med., 2009, 15, 293-299) using combinations ofthe following fluorochrome-conjugated mAbs: SP34-2 (CD3; Pacific Blue,Alexa700), L200 (CD4; AmCyan, BV510), SK-1 (CD8a; PerCP-Cy5.5), MAB11(TNF-α; FITC, PE), B27 (IFN-γ; APC), FN50 (CD69; PE, PE-TexasRed), B56(Ki-67; FITC), and in polycytokine analyses, JES6-5H4 (IL-2; PE, PECy-7). To determine the cell surface phenotype of Mtb-specific CD8+ Tcells, mononuclear cells were stimulated as described above, except thatthe CD28 co-stimulatory mAb was used as a fluorochrome conjugate toallow CD28 expression levels to be later assessed by flow cytometry, andin these experiments, cells were surface-stained after incubation forlineage markers CD3, CD4, CD8, CD95 and CCR7 (see below for mAb clones)prior to fixation/permeabilization and then intracellular staining forresponse markers (CD69, IFN-γ, TNF-α). Data was collected on an LSR-II(BD Biosciences). Analysis was performed using FlowJo software (TreeStar). In all analyses, gating on the lymphocyte population was followedby the separation of the CD3+ T cell subset and progressive gating onCD4+ and CD8+ T cell subsets. Antigen-responding cells in both CD4+ andCD8+ T cell populations were determined by their intracellularexpression of CD69 and one or more cytokines (either or both of theIFN-γ and TNF; ±IL-2 in polycytokine analyses). After subtractingbackground, the raw response frequencies were memory corrected, aspreviously described (Hansen et al., Nature, 2011, 473, 523-527 andHansen et al., Nat. Med., 2009, 15, 293-299) using combinations of thefollowing fluorochrome-conjugated mAbs to define the memory vs. naivesubsets SP34-2 (CD3; Alexa700, PerCP-Cy5.5), L200 (CD4; AmCyan), SK-1(CD8a; APC, PerCP-cy-5.5), MAB11 (TNF-α; FITC), B27 (IFN-γ; APC), FN50(CD69; PE), CD28.2 (CD28; PE-TexasRed), DX2 (CD95; PE), 15053 (CCR7;Pacific Blue), and B56 (Ki-67; FITC). For memory phenotype andpolycytokine analysis of Mtb Ag-specific T cells, all cells expressingCD69 plus one or more cytokines were first Boolean gated, and then thisoverall Ag-responding population was subdivided into the subsets ofinterest on the basis of surface phenotype or cytokine productionpattern.

Mycobacterial Culture:

Tissues routinely collected at necropsy for Mtb burden analysis in bothStudy 3 and 4 included: 30 stereologic punches from right lung lobes, 10punches from left lung lobes, trachea, left hilar LN, right hilar LN,left carinal LN, right carinal LN, paratracheal LN, mediastinal LN,axillary LN, inguinal LN, mesenteric LN, spleen, pancreas, left mediallobe of liver, right medial lobe of liver, left lateral lobe of liver,right lateral lobe of liver, liver caudate, left kidney, and rightkidney. In Study 4, retropharyngeal LN, tonsil, submandibular LN, andiliosacral LN were also collected and cultured. In one RM in Study 4,mycobacterial culture analysis was not reported due to grosscontamination of thoracic tissues with granuloma contents. Tissues werecollected in HBSS and were then homogenized in an IKA grinder tube witha IKA Ultra-Turrax Tube Drive homogenizer. The tissue homogenate wasthen filtered over a 70 μm wire screen to remove debris and 200 μl ofthis material was plated neat and in serial dilutions (1/10, 1/100) on7H11 agar plates (Remel). All plates were incubated at 37° C. and M.tuberculosis growth was enumerated 28 and 42 days later. Bacterialburden was calculated in CFU per gram of tissue. A tissue was consideredMtb+ if any colonies with the correct morphologic features wereidentified. Selected cultures were analyzed by the Ziehl-Neelsen methodfor acid-fast bacteria to confirm colony morphologic features.

Data Preparation for Statistical Analysis:

Three outcome measures were evaluated for evidence of a differenceacross treatment arms within each study: CT scan area-under the curvefrom challenge to day 112 post-challenge, pathologic score at necropsy,and Mtb culture at necropsy. The equivalence of the three Study 4vaccine groups by these outcome measures (see, FIG. 49) justified apooled analysis. An outcome measure that combines necropsy score andculture results into a single value that can be evaluated across bothefficacy studies was evaluated. Associations between these outcomes andMtb-specific T cell responses measured by ICS were assessed.

Area Under the Log CT Scan-Determined Pulmonary Disease Volume Curve:

The AUC of the log-transformed CT scan-determined pulmonary diseasevolume measurements from time 0 (set to 0) to day 112 was computed.Missing values for monkeys taken to necropsy before the full series ofscheduled CT scan time points were imputed. The AUC of this augmenteddata was computed. The imputation procedure that was employed usedlinear regression to estimate missing values from previous time points.As a sensitivity analysis, the missing values were imputed using a moreconservative rule (replacing missing values with the largest non-missingvalue at the same time point among monkeys receiving the same treatment,excluding for further conservatism one high-valued outlier unvaccinatedRM); the resulting AUCs were highly insensitive to this procedure(Pearson correlation over 0.99 for both studies).

Necropsy Score Data:

The non-negative count valued necropsy scores are amenable to Poissonmodeling. Model evaluations supported inclusion of the additionalparameter for overdispersion in the negative binomial model. In TB Study4, the estimated extra parameter was zero, so this model was equivalentto a simple Poisson model. For this Poisson model, a sandwich-basedestimates of variance-covariance matrices was employed as an alternativemethod to account for overdispersion, using the vcovHC function in thesandwich package (Zeileis, J. Stat. Software, 2004, 11, 1-17 andZeileis, J. Stat. Software, 2006, 16, 1-16) in R.

Necropsy Culture Data:

Necropsy culture inputs were quantitative measures of culture growthwith multiple replicates per tissue. These data were treated as binaryindicators of a culture being positive versus negative (zero), and thetotal number of positive cultures was evaluated. Model evaluations ofnecropsy culture outcome data favored the more expressive negativebinomial models over Poisson models, and did not support using the ZIPmodel. One animal in TB Study 4 (Rh30072) was missing necropsy culturedata but did have necropsy score data; the analyses of the culture datatherefore excluded this RM, but as described below, the missing valuefor use in computing the combined scaled outcome measure was imputed.

Combined Scaled Outcome Measure:

As shown in FIG. 50, a strong correlation between the necropsy score andnecropsy culture outcome measures within each study was observed,although the scales were different: necropsy cultures are about onethird as large as necropsy scores, across both studies. Theintermediate, study-specific combined scaled outcome measure was createdto gain measurement precision by averaging these two very similaroutcome measures. To ensure that each receives equal weight in thecombined measure, and to maintain discreteness in support of Poissonanalysis of the statistic, the inputs were scaled by multiplying thenecropsy culture values by 3 and then adding these to the necropsyscores. For Study 4, one RM (I3) had a missing necropsy culture value.For this animal, the combined scaled outcome measure was computed usingan imputed necropsy culture value, which was obtained by multiplying theobserved necropsy score value by the estimated coefficient from a simplelinear regression model relating the two values. This monkey's necropsyscore value was 29, its imputed necropsy culture value was 8, and itscombined scaled outcome measure value was 53. A negative binomialregression model of these study-specific combined measures versustreatment and study was employed, and used the estimated coefficient onstudy (0.3796) to further scale the TB Study 3 combined scaled outcomevalues. Therefore, the combined scaled outcome variable for TB Study 3RM is the study-specific value multiplied by 0.3796 and then rounded tomaintain the discreteness of the final variable for Poisson analysis.

End of Vaccine Phase T Cell Response Data:

Longitudinal flow cytometric ICS measures of CD4+ and CD8+ immuneresponses targeting the 9 individual genes in the RhCMV vaccines, andCFP10 was evaluated. The primary summary of these data was a measure ofthe immune response at the end of vaccine phase. These pre-challengebaseline immunogenicity values are geometric means of three independentmeasurements over the time periods shown in FIG. 42 (panel a) and FIG.44 (panel a). Totals over 6 or 9 antigens were computed prior tolog-transformation and normalization. Normalization shifts and scalesthese values to have mean zero, standard deviation 1 so that units havethe interpretation of z-scores measuring the number of standarddeviations an immune measurement (on the log scale) is from the overall(study-specific) mean of that measurement.

Statistical Analysis:

All statistical analyses were conducted in R40.

Efficacy:

Non-parametric tests were employed for primary comparisons andparametric models were used for estimating confidence intervals oftreatment effects. For comparisons of outcome measures across pairs ofgroups, we used two-sided Wilcoxon tests. Boxplots show unadjustedp-values of only the pairwise comparisons that are significant at the0.05 level. Holm adjustment was employed for the specified primarynon-parametric comparisons: between unvaccinated and vaccinated TB Study3 groups, and separately between BCG-only and other vaccinated groups.For TB Study, 4 similarly applied Holm-adjustment within groups ofcomparisons between unvaccinated and vaccinated TB Study 4 groupsindividually, and between the original 68-1 9Ag vector and the twomodifications (68-1 6Ag, and 68-1.2 9Ag). Boxplots show unadjustedp-values; Holm-adjusted p-values are shown in FIG. 51. Vaccine efficacyis reported as 100%−W, where W is 100 times the estimated rate (orconfidence limit) of the Poisson or negative binomial model representinga count-valued outcome measure (necropsy score, necropsy culture, or thecombined scaled outcome measure) among a vaccine group, as a fraction ofthe rate for Unvaccinated RM. For analysis of correlations acrossoutcome variables, Spearman's rank-transformed correlation statistic (r)and test were used. For Spearman's test, p-values were computed via theasymptotic t approximation using the cor.test method in R.

Immunogenicity and Correlates Analysis:

For comparisons of immunogenicity across vaccine-receiving treatmentgroups at pre-challenge baseline, Kruskal-Wallis (KW) tests wereemployed. The boxplots indicate significance of pairwise Wilcoxon testsif both KW and Wilcoxon tests had unadjusted p-values ≤0.05. Due to themissing of blood immunology data from week 41 of RM U6 (Study 3;BCG-only group), for FIG. 42 (panel d), n=6 for the BCG group; and forFIG. 42 (panel c), the data point for the data point for this RM usedonly 2, rather than 3, time points for averaging. For analysis of immuneresponse correlations with the combined scaled outcome measure (definedabove) among RM receiving RhCMV and no BCG, Spearman's statistic andtest was computed, and the scatterplots also show the curve of thebest-fitting negative binomial model. The low correlations (see, FIG.55) were confirmed through sensitivity analyses that also found in eachstudy separately and in each outcome measure, so the lack ofsignificance is not an artifact of these analysis choices. Extensivenon-parametric analyses as well as parametric analyses employingnegative binomial models to estimate single-parameter andmulti-parameter associations between this combined scaled measure andpre-challenge immune responses to RhCMV immunogens revealed nostatistically supported immune correlates of that outcome, or of otherrelated outcomes.

Example 5: Studies #3 and #4

To initially test the hypothesis that TEM responses elicited by RhCMV/TBvectors would manifest a higher efficacy than BCG, 3 groups of RM (n=7each; all naturally RhCMV-infected at study assignment) were vaccinatedwith: 1) RhCMV/TB vectors alone (a set of 4 RhCMV vectors based on the68-1 strain that together express 9 different Mtb proteins: ESAT-6,Ag85A, Ag85B, Rv3407, Rv1733, Rv2626, Rpf A, Rpf C, Rpf D; see, FIG. 46,panel a), 2) BCG alone, and 3) BCG followed by RhCMV/TB, according tothe protocol outlined in FIG. 42, panel a. As expected, RhCMV/TB vectorselicited and maintained high frequency CD4+ and CD8+ T cell responses inblood to all 9 Mtb inserts, as measured by overlapping 15-mer peptidemix-induced expression of intracellular TNF and/or IFN-γ by flowcytometric intracellular cytokine (ICS) analysis, and in plateau phase,these responses were predominantly effector differentiated, manifestingeither a fully differentiated TEM phenotype (CD8+) or a mixedtransitional and fully differentiated TEM phenotype (CD4+) (see, FIG.42, panels b-d). About half of the RhCMV/TB-elicited, Mtb Ag-specificCD4+ and CD8+ T cells responding in the ICS assays produced both TNF andIFN-γ (with or without IL-2) with the remainder predominantly producingTNF alone (see, FIG. 42, panel e). BCG elicited circulating CD4+ andCD8+ T cell responses to 8 of the 9 insert Ags (all except ESAT-6, whichis not expressed by BCG22). These responses predominantly manifested acentral memory phenotype for the CD4+ T cells and a fully differentiatedTEM phenotype for the CD8+ T cells. However, in peripheral blood, theoverall magnitude of the BCG-elicited T cell responses to these Ags wasconsiderably less (5-10-fold) than in RhCMV/TB-vaccinated RM, and themajority of these T cells produced either TNF or IL-2 alone (CD4+) orTNF or IFN-γ alone (CD8+), but not both TNF and IFN-γ (see, FIG. 42,panels b-e). Indeed, the BCG-induced CD4+ and CD8+ T cell response tothese Ags was not large enough to measurably change the plateau-phasemagnitude, phenotype and function of the TB Ag-specific responses in theRM that received both BCG and RhCMV/TB relative to the RM that receivedRhCMV/TB vaccination alone (see, FIG. 42, panels b-e). Differences inresponse magnitude between BCG- and RhCMV/TB-vaccinated RM were lessapparent in bronchoalveolar lavage (BAL) fluid, with the responses inthe latter group only marginally higher than in the former group (see,FIG. 42, panel f).

Referring to FIG. 42, the immunogenicity of RhCMV/TB and BCG vaccines inStudy 3 is shown. Panel a is a schematic of the vaccination andchallenge protocol and RM groups of Study 3. Panel b shows alongitudinal analysis of the overall CD4+ and CD8+ T cell response tothe 9 Mtb insert proteins after vaccination with the designatedvaccines. The background-subtracted frequencies of cells responding withTNF and/or IFN-γ production by flow cytometric ICS assay to peptidemixes comprising each of the Mtb proteins within the memory CD4+ or CD8+T cell subset were summed with the figure showing the mean (±SEM) ofthese overall (summed) responses at each time point. Panel c showsboxplots comparing the individual Mtb protein (each of the 9 Mtb insertsplus the non-insert CFP-10)-specific and overall (summed) Mtb-specificCD4+ and CD8+ T cell response frequencies (defined by TNF and/or IFN-γproduction) in peripheral blood between the vaccine groups at the end ofthe vaccine phase (each data point is the mean of response frequenciesin 3 separate samples from weeks 44-49; indicates no response detected).Penal d shows boxplots comparing the memory differentiation of thevaccine-elicited CD4+ and CD8+ memory T cells in peripheral bloodresponding to Ag85A with TNF and/or IFN-γ production at the end ofvaccine phase (week 47). Memory differentiation state was based on CD28vs. CCR7 expression, delineating central memory (TCM), transitionaleffector memory (TTREM), and effector memory (TEM), as designated. Penale shows boxplots comparing the frequency of vaccine-elicited CD4+ andCD8+ memory T cells in peripheral blood responding to Ag85A with TNF,IFN-γ and IL-2 production, alone and in all combinations at the end ofvaccine phase (week 49). Panel f shows boxplots comparing the individualMtb protein-specific and overall (summed) Mtb-specific CD4+ and CD8+ Tcell response frequencies (defined by TNF and/or IFN-γ production) inbronchoalveolar lavage (BAL) fluid between the vaccine groups at the endof the vaccine phase (weeks 46-47; indicates no response detected). Inpanels c-f, the Kruskal-Wallis (KW) test was used to determine thesignificance of differences between vaccine groups with the Wilcoxonrank sum test used to perform pair-wise analysis if KW p values were≤0.05; brackets indicate pair-wise comparisons with Wilcoxon p values≤0.05.

Referring to FIG. 46, a description of RhCMV/TB vectors is shown. Panela is a diagram showing the insertion sites for TB Ag cassettes in eitherthe RhCMV 68-1 and 68-1.2 BAC backbones. The top 4 constructs useexogenous promoters (either MCMV IE or EF1a) to drive insert expression,whereas the bottom construct (68-1 RhCMV/TB-6Ag) replaces the Rh107 openreading frame with the 6 Ag insert, and relies upon the endogenous Rh107promoter to regulate insert expression. Panel b shows a RT-PCR analysisof the 68-1 RhCMV/TB-6Ag vector confirming deletion of Rh107,concomitant expression of the 6 Ag TB insert and unchanged expression ofboth IE1 and the adjacent Rh108 open reading frame. In this experiment,telomerized rhesus fibroblasts (TRF) were infected with the vector at amultiplicity of infection (MOI) of 3 and RNA was harvested and cDNAgenerated at 48 hours post-infection. RT-PCR was performed using primersspecific for internal regions of the indicated genes to demonstratedeletion of Rh107 and expression of the 6Ag insert as well assurrounding open reading frames. Panel c shows a Western Blot analysisof the HA-tagged 6 Ag insert expression (arrow) by the 68-1 RhCMV/TB-6Agvector. TRFs were infected with RhCMV 68-1 or the 68-1 RhCMV/TB-6Agvector (RhCMV 68-1 ΔRh107-TB6Ag) at an MOI=3, harvested at fullcytopathic effect, and subjected to western blotting directed at the HAtag. HeLa cells transfected with a plasmid expressing the 6 Ag insert(pOri-TB6Ag) are shown as a control.

Fifty weeks after initial vaccination, the 3 groups of vaccinated RM anda control group of unvaccinated RM (n=8; also naturally CMV+) werechallenged by intrabronchial instillation of 25 colony-forming units(CFUs) of Erdman strain Mtb bacteria into the right lower lobe. Theeffectiveness of challenge was confirmed by de novo development of CD4+and CD8+ T cell responses to the CFP-10 Ag in all RM (see, FIG. 43,panel a, and FIG. 47, panel a; the Mtb-expressed CFP-10 Ag was notincluded in the RhCMV/TB vectors, and, like ESAT-6, is not expressed byBCG22). The development of pulmonary disease after challenge wasmonitored every two weeks by CT scan assessment of lesional volume, butprimary outcome was determined by pathologic examination (pathologicscore; see, FIG. 48) and by extensive mycobacterial culture of lung(sampled using stereology), as well as lung-draining and other chestlymph nodes (LNs), peripheral LNs and selected organs (spleen, liver,kidney, pancreas) at necropsy (see Methods), with necropsy performedeither at clinical endpoint, or after 20 weeks post-infection (pi), byrandomization (see, FIG. 42, panel a). Pulmonary disease developedrapidly in unvaccinated control RM with progression to severe (>10,000mm³) lung parenchymal disease by CT scan in 7/8 RM by day 56 pi and allRM by day 98 pi (see, FIG. 43, panel b). In both RM groups that receivedBCG, the development of pulmonary disease was more variable, but 5 of 7RM in each group developed severe disease by day 98 pi. In contrast, 5of 7 of the RM vaccinated with RhCMV/TB vectors alone developed onlymild pulmonary disease (<3,000 mm³, n=4) or no disease (n=1), and theoverall area-under-the-curve (AUC) of pulmonary lesion volume of thisgroup during the first 16 weeks pi was significantly reduced from theunvaccinated control group (see, FIG. 43, panel c, and FIG. 49, panela).

Referring to FIG. 43, the outcome of Mtb challenge (Study 3) is shown.Panel a shows the development of peripheral blood CD4+ T cell responsesto the peptide mixes comprising the non-vaccine insert Mtb proteinCFP-10 in all Study 3 RM after Mtb challenge by flow cytometric ICSanalysis (response defined by TNF and/or IFN-γ production afterbackground subtraction in memory subset; CFP-10-specific CD8+ T cellresponses shown in FIG. 47, panel a). Panel b shows CT quantification ofdisease volume in the pulmonary parenchyma after Mtb challenge (presenceor absence of draining LN enlargement indicated by closed vs. opensymbols). Panel c shows boxplots comparing the AUC of CT-determinedpulmonary lesional volume (day 0-112) of the 4 RM groups. Panels d-eshow boxplots comparing the extent of TB at necropsy measured by Mtbrecovery with mycobacterial culture and by pathologic disease score inlung parenchyma (panel d), all non-lung parenchymal tissues (panel e)and all tissues (panel f). In panels c-f, unadjusted Wilcoxon p values≤0.05 are shown (see, FIG. 49, panel a).

Referring to FIG. 47, the development of de novo Mtb-specific CD8+ Tcell responses after Mtb challenge is shown. Panels a and b show thedevelopment of peripheral blood CD8+ T cell responses to the peptidemixes comprising the non-vaccine insert Mtb protein CFP-10 in Study 3(panel a) and Study 4 (panel b) RM after Mtb challenge by flowcytometric ICS analysis (response defined by TNF and/or IFN-γ productionafter background subtraction in memory subset). CorrespondingCFP-10-specific CD4+ T cell responses are shown in FIG. 43, panel a andFIG. 45, panel a. All Mtb-challenged RM, including vaccinated RM thatdid not manifest post-challenge disease, showed multiple post-challengesamples with above-threshold CFP-10-specific CD4+ and CD8+ T cellresponses, indicating a de novo response to challenge. Panel c shows thedevelopment of peripheral blood CD8+ T cell responses to the peptidemixes comprising the Ag85B, Rv1733 and Rpf proteins in Study 4, group 3RM, who received the RhCMV/TB-6Ag vaccine lacking these insert Ags(corresponding CD4+ T cell responses shown in FIG. 45, panel b). Again,de novo post-challenge induction of responses to these 3 Mtb proteinswas observed in all group 3 RM.

Referring to FIG. 48, the pathologic scoring of TB disease at necropsyis shown. The tables show the criteria and scoring system used toquantify the pathologic extent of TB disease at necropsy in Study 3 and4 RM. Note that every RM receives a separate score for 7 lung lobes,chest wall, each separate lymph node group (both chest and non-chest),liver, spleen, and each other involved organ, with the sum of thesescores being the overall Pathologic Score. The sum of the scores for the7 lung lobes is the Lung Pathologic Score, with the sum of all otherscores (including chest wall and lymph nodes) being the Non-LungPathologic Score. The sum of all scores (Lung and Non-Lung) is theOverall Pathologic Score.

Referring to FIG. 49, a summary of outcome statistics is shown. Panels aand b show Study 3 analysis. Panels c and d show Study 4 analysis. Panele shows the combined analysis.

The CT-determined lesional AUC through week 16 pi closely correlatedwith pulmonary parenchymal disease at necropsy as measured by bothpathologic scoring and mycobacteria culture (see, FIG. 50, panel a), andas such, the extent of lung disease at necropsy was significantlyreduced in the RhCMV/TB vector group compared to unvaccinated controlsby both these measures (see, FIG. 43, panel d, and FIG. 49, panel a). Nosignificant pulmonary disease reduction was observed in the BCG-onlyvaccinated cohort by either necropsy measure, and the BCG+RhCMV/TBvaccine regimen resulted in only a modest reduction in mycobacterialburden. TB is typically not restricted to pulmonary parenchyma in RMgiven this dose of Erdman strain Mtb and necropsy analysis revealedextensive extra-pulmonary disease in the unvaccinated RM, including bothlung-associated lymph node involvement and extra-thoracic spread (see,FIG. 51). The extent of disease as measured by pathologic score wasclosely correlated with the extent of disease measured by mycobacterialculture (see, FIG. 50, panel b), and by both criteria, extra-pulmonarydisease was dramatically reduced in the RhCMV/TB-vaccinated cohort,resulting in a significant reduction in the overall extent of disease inthis cohort relative to unvaccinated RM (see, FIG. 43, panels e and f;FIG. 49, panel a; and FIG. 51). By Poisson modeling, the overall extentof disease was reduced in the RhCMV/TB-vaccinated group by 68.7% bymycobacterial culture (P<0.0001) and 67.3% by pathologic score(P<0.0001) relative to unvaccinated controls (see, FIG. 49, panel b). Incontrast, the extent of extra-pulmonary and overall disease inBCG-only-vaccinated RM was not significantly different from unvaccinatedRM by either criteria, and in keeping with this, the extent ofextra-pulmonary and overall disease in the RM given the RhCMV/TB vaccinealone was also significantly reduced from the BCG-only-vaccinated RM.Using the same Poisson modeling, RhCMV/TB vaccination reduced overalldisease relative to BCG vaccination by 57.7% (P=0.0007) and 51.4%(P=0.01) for mycobacterial culture and pathologic score, respectively(see, FIG. 49, panel b). Pre-vaccination of RM with BCG 6 weeks prior toinitial RhCMV/TB vaccination appeared to substantially reduce theextra-pulmonary, as well as pulmonary, efficacy of RhCMV/TB vaccinationalone, as the BCG+RhCMV/TB-vaccinated group showed only a very modestreduction in mycobacterial recovery in all sites relative tounvaccinated controls (see, FIG. 43, panels d-f; FIG. 49, panel a; andFIG. 51).

Referring to FIG. 50, a comparison of different measures of TB infectionoutcome is shown. Panels a and b show the correlation of pulmonaryparenchymal disease as measured by CT scan-determined disease volume(AUC through day 112) post-infection vs. Mtb culture of lung samples(panel a) and lung pathologic score (panel b) at necropsy of Study 3 RM.Panel c shows the correlation of overall Mtb culture vs. overallpathologic score at necropsy of Study 3 RM. Panels c and d show the sameanalysis for Study 4 RM. Spearman correlation coefficients (r) andassociated p values are shown in each plot.

Referring to FIG. 51, a summary of TB disease outcome at necropsy ofStudy 3 is shown. The lung, non-lung, and overall extent of Mtb diseaseby mycobacterial culture (# positive cultures; left y axis) andpathologic scoring (right y-axis; see FIG. 48) is shown for eachindividual Study 3 RM.

To confirm and further characterize RhCMV/TB efficacy, a second, largerMtb challenge study (n=9 RM per group; all RhCMV+ at assignment) wasperformed using a lower dose of Erdman strain and in which we comparedthe same 68-1 RhCMV/TB-9 Ag vaccine used in Study 3 (group 1) with ananalogous RhCMV/TB-9 Ag) vaccine based on the 68-1.2 vector backbone (inwhich repaired expression of Rh157.5 and Rh157.4 results in distinctCD8+ T cell epitope targeting; group 2), and a single 68-1 RhCMV/TB-6 Agvector expressing a 6-Ag Mtb polyprotein (Ag85A; ESAT-6; Rv3407; Rv2626;Rpf A; Rpf D) (group 3) (see, FIG. 44, panel a; FIG. 46). 68-1 RhCMVvectors elicit unconventional CD8+ T cell responses that are restrictedby MHC-II and MHC-E, whereas the CD8+ T cell responses elicited by68-1.2 RhCMV vectors are conventionally MHC-Ia restricted; thus, thegroup 1 vs. group 2 comparison allows determination of the contributionof unconventionally restricted CD8+ T cells to RhCMV/TB efficacy. In thegroup 1 vs. group 3 comparison, whether the efficacy observed with the 4RhCMV/TB vector set encoding 9 Mtb Ags (3 each in the acute phase,latency and resuscitation Ag types) can be recapitulated by a singleRhCMV/TB vector expressing 6 Mtb Ags (2 each from these Ag types) whichis more appropriate for clinical translation was determined. Themagnitude of the overall Mtb-specific and individual Mtb insert-specificCD4+ and CD8+ T cell responses elicited by the 68-1 and 68-1.2RhCMV/TB-9 Ag vectors were comparable in blood throughout thevaccination phase, and in BAL and lymph node at the end of vaccinationphase, as was the memory differentiation and functional phenotype of theMtb-specific response in blood (see, FIG. 44, panels b-g). However, theCD8+ T cells elicited by the 68-1 RhCMV/TB vaccine were unconventionally(MHC-II and MHC-E) restricted, whereas those elicited by 68-1.2 RhCMV/TBvaccine were conventionally (MHC-Ia) restricted (see, FIG. 52). Theobserved immune responses against the 6 Ags common to both 68-1RhCMV/TB-6 Ag and RhCMV/TB-9 Ag vaccines are similar between groups 1and 3 with respect to magnitude, phenotype and (unconventional) MHCrestriction, except for slightly different levels of CD8+ T cellresponses in LN (see, FIG. 44, panels b-g; FIG. 52).

Referring to FIG. 3, the immunogenicity of RhCMV/TB vaccines (Study 4)is shown. Panel a shows a schematic of the vaccination and challengeprotocol and RM groups of Study 4. Panel b shows a longitudinal analysisof the overall CD4+ and CD8+ T cell response to the 9 Mtb Ags aftervaccination with the designated vaccines, as described in FIG. 42, panelb. Panel c shows boxplots comparing the individual Mtb protein (each ofthe 9 Mtb inserts plus the non-insert CFP-10)-specific and overall(summed) Mtb-specific CD4+ and CD8+ T cell response frequencies (definedby TNF and/or IFN-γ production) in peripheral blood between the vaccinegroups at the end of the vaccine phase (each data point is the mean ofresponse frequencies in 3 separate samples from weeks 49-55; indicatesno response detected). Panel d shows boxplots comparing the memorydifferentiation (see, FIG. 42, panel d) of the vaccine-elicited CD4+ andCD8+ memory T cells in peripheral blood responding to Ag85A with TNFand/or IFN-γ production at the end of vaccine phase (weeks 51-52). Panele shows boxplots comparing the frequency of vaccine-elicited CD4+ andCD8+ memory T cells in peripheral blood responding to Ag85A with TNF,IFN-γ and/or IL-2, alone and in all combinations at the end of vaccinephase (weeks 49-50). Panels f and g show boxplots comparing theindividual Mtb protein (the 9 Mtb inserts plus the non-insertCFP-10)-specific and overall (summed) Mtb-specific CD4+ and CD8+ T cellresponse frequencies (defined by TNF and/or IFN-γ production) in BAL(panel f) and in peripheral LN (panel g) between the vaccine groups atthe end of vaccine phase (weeks 46-47; indicates no response detected).In panels c-g, statistics performed as described in FIG. 42 withbrackets indicating pair-wise comparisons with Wilcoxon p values ≤0.05.

Referring to FIG. 52, the MHC-restriction analysis of RhCMV/TBvector-elicited CD8+ T cell responses in Study 4 is shown. RhCMV/TBvaccine-elicited CD8+ T cells were epitope-mapped in representativegroup 1, 2 and 3 RM from Study 4 using flow cytometric ICS to detectrecognition of each consecutive, overlapping 15-mer gag peptidecomprising the indicated TB proteins. Peptides resulting in specificCD8+ T cell responses are indicated by a box, with the color of the boxdesignating MHC restriction as determined by blocking with theanti-pan-MHC-I mAb W6/32, the MHC-E blocking peptide VL9 and the MHC-IIblocking peptide CLIP. The epitope restriction profiles of the strain68-1 RhCMV/Ag85B/ESAT-6- and RhCMV/Rpf A/Rpf C/Rpf D-elicited CD8+responses to Rpf A, Ag85B, and ESAT-6 are produced here for comparisonwith 68-1.2 versions of these vectors and for Rpf A and ESAT-6, with the68-1 RhCMV/TB-6Ag vector.

After a 56 week vaccination period, all 27 vaccinated RM in groups 1-3and 9 RhCMV+ unvaccinated control RM (group 4) were intrabronchiallychallenged with 10 CFUs of Erdman strain Mtb bacteria—the reduction indose relative to Study 3 intended to slow TB progression in Study 4 RMto more closely resemble the course of human Mtb infection. In addition,post-challenge BAL was not performed in this experiment to preventprocedure-related mortality or enhancement of bacteria spread within thelung. All RM developed de novo CFP-10-specific T cell responses in bloodfollowing challenge, and the RhCMV/TB-6 Ag-vaccinated RM (group 3) alsodeveloped de novo T cell responses in blood to the Ag85B, Rpf C, andRv1733 Ags, which were not included in their vaccine (see, FIG. 45,panels a and b; FIG. 47, panels b and c). All 9 unvaccinated (group 4)RM developed TB lesions on CT scans by day 28 pi, but, as anticipated,the disease progression in this study was slower than in Study 3 (see,FIG. 45, panel c), and only 2 unvaccinated (group 4) control RMdeveloped endpoint TB disease over the course of observation (see, FIG.44, panel a). Remarkably, 13 of the 27 vaccinated RM (5 each in group 1and group 3; 3 in group 2) did not develop any radiologic signs ofpulmonary TB (including no hilar adenopathy) at any time point throughto random elective necropsy at >16 weeks pi, and the averageCT-determined lesional AUC in lung parenchyma of the overall cohort ofvaccinated RM was significantly reduced from the unvaccinated group(see, FIG. 45, panels c and d; FIG. 49, panel c). At necropsy, none ofthe 13 CT-negative RM from vaccine groups 1-3 manifested any macroscopicgranulomatous disease, and 10 of these 13 were culture negative in alltissues (the remaining 3 were Mtb+ in lung-draining lymph nodes; see,FIG. 50, panels c and d; FIG. 54). Despite the development of CD4+ andCD8+ T cell responses to Mtb proteins not in their vaccine in lung,lung-draining and peripheral lymph nodes, and spleen (see, FIG. 53),histopathologic examination of lung and lung-draining lymph nodesections in these 13 TB disease-free RM showed no granulomatousinflammation. The overall extent of disease by mycobacterial culture andpathologic score was strongly correlated in this experiment (see, FIG.50, panel d), and was significantly reduced in the overall (pooledgroups 1-3) RhCMV/TB vaccinated group compared to the unvaccinatedcontrol group, with no significant difference in efficacy betweenindividual groups 1-3, and similar protection in both lung and non-lungtissues (see, FIG. 45, panels e-g; FIG. 49, panel c; FIG. 54). For thepooled vaccinated group, the overall reduction in disease extentrelative to the unvaccinated control group was 74.5% by mycobacterialculture (P=0.0024) and 61.4% by pathologic score (P=0.0011) usingPoisson modeling (see, FIG. 51). The finding of efficacy with 68-1.2RhCMV/TB vaccination indicates that efficacy is not dependent onunconventional MHC-II and MHC-E-restricted CD8+ T cells, indicating thatprotection can be mediated by either conventional or unconventional CD8+T cells, or is independent of CD8+ T cells altogether.

Referring to FIG. 45, the outcome of Mtb challenge (Study 4 and Overall)is shown. Panels a and b show the development of peripheral blood CD4+ Tcell responses to the peptide mixes comprising the non-vaccine insertMtb protein CFP-10 in all Study 4 RM (panel a), and comprising theAg85B, Rv1733 and Rpf proteins in group 3 RM only (panel b; RMvaccinated with the single 68-1 RhCMV/TB-6Ag vector lacking these 3inserts) after Mtb challenge by flow cytometric ICS analysis, asdescribed in FIG. 43, panel a (peripheral blood CD8+ T cell responsesand tissue CD4+ and CD8+ T cell responses to these same Ags are shown inFIG. 47, panels b and c and FIG. 53, respectively). Panel c shows CTquantification of disease volume in the pulmonary parenchyma after Mtbchallenge (presence or absence of draining LN enlargement indicated byclosed vs. open symbols). Panel d shows boxplots comparing the AUC ofCT-determined pulmonary lesional volume (day 0-112) of the unvaccinatedRM vs. all RhCMV/TB-vaccinated RM vs. RM in each individual RhCMV/TBvaccine group. Panels e-g show boxplots comparing the extent of TB atnecropsy measured by Mtb recovery with mycobacterial culture and bypathologic disease score in lung parenchyma (panel e), all non-lungparenchymal tissues (panel f) and all tissues (panel g) in the same RMgroups. Panel h shows a boxplot comparing the outcome of Mtb challengein all unvaccinated RM vs. all RhCMV/TB-only vaccinated RM across bothStudies 3 and 4 using a scaled outcome measure that combines bothmycobacterial culture and pathologic score data. In panels d-h,unadjusted Wilcoxon p values ≤0.05 are shown (see, FIG. 49, panel c).

Referring to FIG. 53, an analysis of non-vaccine-elicited, Mtb-specificCD4+ and CD8+ T cell responses at necropsy (response to Mtb challenge)is shown. Panel a shows flow cytometric ICS analysis demonstratingperipheral blood and tissue CD4+ and CD8+ T cell responses to thepeptide mixes comprising the non-vaccine insert Mtb protein CFP10 in the1 Study 3 RM and the 13 Study 4 RM (5, 3, and 5 from Groups 1, 2 and 3,respectively) without pathologic evidence of TB disease at necropsy(see, FIGS. 50 and 54; response defined by TNF and/or IFN-γ productionafter background subtraction in memory subset). Panel b shows a similaranalysis of T cell responses to peptide mixes comprising the Ag85B,Rv1733 and Rpf proteins in the 5 Study 4, group 3 RM (vaccinated withthe RhCMV/TB-6 Ag vaccine lacking these inserts) who failed to manifestTB disease after challenge. These data confirm that these protectedmonkeys were sufficiently exposed to Mtb infection after challenge todevelop a robust systemic response to TB proteins that were not presentin their vaccine.

Referring to FIG. 54, a summary of outcome at necropsy of Study 4 isshown. The lung, non-lung, and overall extent of Mtb disease bymycobacterial culture (#positive cultures; left y axis) and pathologicscoring (right y-axis; see, FIG. 48) are shown for each individual Study4 RM.

It is remarkable that despite the fact that the average extent of TBprogression in the unvaccinated control monkeys in Studies 3 and 4 wasquite different, the reduction in disease with RhCMV/TB vaccination wassimilar in both studies. Indeed, using a normalized, combination outcomeparameter based on both mycobacterial culture and pathologic score, itwas estimated that across both studies the extent of disease in theRhCMV/TB-vaccinated RM was reduced 68% relative to unvaccinated controls(P=0.0019) (see, FIG. 45, panel h; FIG. 51). In contrast to the “all ornone” efficacy of the RhCMV/SIV vaccine against rapidly progressive SIVinfection, the protection afforded by RhCMV/TB against the more slowlyprogressive Mtb infection appears to be graded, including RM withapparent sterilizing protection, RM with no macroscopic disease but withvery focal bacterial persistence, and a higher fraction of RM withreduced progression compared to unvaccinated controls (see, FIGS. 50 and54). However, there are also vaccinated RM that developed progressiveand ultimately fatal TB disease similar to the unvaccinated controls.This outcome heterogeneity was not predicted by the RhCMV/TB-elicited,TNF/IFN-γ-defined, CD4+ or CD8+ T cell response magnitudes in blood, BALor LN prior to challenge (see, FIG. 55). The observation that BCGvaccination 6 weeks prior to RhCMV/TB vaccination reduces efficacyalmost 1 year later supports the concept that mycobacteria-inducedimmune responses do include an anti-protective component.

Referring to FIG. 55, immune correlates analysis of Studies 3 and 4 areshown. Panels a and b show the relationship between the end-of-vaccinephase overall TB-specific CD4+ and CD8+ T cell responses in peripheralblood (panel a) and BAL (panel b) to the same scaled outcome measurethat combines both mycobacterial culture and pathologic score data atnecropsy used in FIG. 45, panel h is shown for all Study 3 and 4 RM thatreceived a RhCMV/TB vaccine only. The T cell responses reflect the sumof responses to the ESAT-6, Ag85A, Ag85B, Rv3407, Rv1733, Rv2626, Rpf A,Rpf C, Rpf D Ags and are presented in units of z-score (number ofstandard deviations that a monkey's normalized total immune response atend of vaccine phase is above the average of the monkeys in that study.Panel c shows the same analysis for overall Mtb-specific CD4+ and CD8+ Tcell responses in peripheral lymph node in RhCMV/TB-vaccinated Study 4RM (lymph node biopsy was not performed in Study 3). Spearmancorrelation coefficients (r) and associated P values are shown in eachplot, with the best-fitting negative binomial curve overlayed Similarresults (lack of significant correlation) were observed for total AUCCD4+ or CD8+ T cell responses for the entire vaccine phase in peripheralblood, and for peripheral blood, BAL, and lymph node responses to onlythe 6 Ags common to all RhCMV/TB-vaccinated RM, or to any individual MtbAg prior to challenge.

These studies show that a parenterally administered RhCMV-based vaccineis able to elicit and maintain over the course of at least a yeareffector responses that can control Mtb at the early stages ofinfection, and that the protection afforded by this vaccine can becomplete, if not sterilizing. To our knowledge, this is the first reportof complete prevention of TB in the RM model. Given TEM responses tonatural, persistent CMV infection are maintained for life, theprotection afforded by this vector platform is likely to be verydurable, probably lifelong. The RhCMV/TB vaccine is efficacious againstaggressive TB in RM and provides treatment of a human CMV/TB vaccinethat would be effective in preventing pulmonary TB in adolescents andadults, and thereby contribute to ending the global TB epidemic.

Over both Studies 3 and 4, RhCMV/TB vaccination reduced the extent ofdisease at necropsy, as measured by both pathologic score and frequencyof mycobacterial culture-positive tissues, by 68% compared tounvaccinated controls (P=0.0019). There was no significant difference inefficacy between cohorts vaccinated with 68-1 vs. 68-1.2 RhCMV vectorbackbones (which differ in CD8+ T cell epitope recognition) or with 68-1RhCMV vectors expressing 9 vs. 6 Mtb proteins. In contrast, BCG was notsignificantly efficacious in this challenge model, and administration ofBCG 6 weeks prior to RhCMV/TB vaccination reduced the efficacy of thelatter vaccine. Across both studies, 14 of the 34 RhCMV/TB-vaccinated RM(41%) showed no granulomatous disease at necropsy (vs. 0 of 17unvaccinated controls; P=0.0018), despite immunologic evidence ofinitial infection after challenge, and 10 of these were mycobacterialculture-negative in all tissues. Thus, the RhCMV/TB vaccine is superiorto BCG in the RM model, and is the first vaccine demonstrated tocompletely prevent progressive TB in primates.

Various modifications of the described subject matter, in addition tothose described herein, will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims. Each reference (including,but not limited to, journal articles, U.S. and non-U.S. patents, patentapplication publications, international patent application publications,gene bank accession numbers, and the like) cited in the presentapplication is incorporated herein by reference in its entirety.

What is claimed is:
 1. A recombinant rhesus cytomegalovirus (RhCMV) orhuman cytomegalovirus (HCMV) vector comprising a nucleic acid sequenceencoding an expressible Mycobacterium tuberculosis (Mtb) antigen,wherein said Mtb antigen is a fusion protein, wherein said fusionprotein is selected from the following two fusion proteins whichcomprise the following Mtb proteins or antigenic fragments thereof inthe order listed: Rv1733-Rv2626c andAg85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.
 2. The recombinant RhCMV or HCMVvaccine vector of claim 1, wherein expression of the Mtb antigen isdriven by an antigen-coding sequence in operable association with apromoter selected from the group consisting of a constitutive CMVpromoter, an immediate early CMV promoter, an early CMV promoter, and alate CMV promoter.
 3. The recombinant RhCMV or HCMV vaccine vector ofclaim 2, wherein the promoter is selected from the group consisting ofEF1-alpha, UL82, MIE, pp65, and gH.
 4. The recombinant RhCMV or HCMVvaccine vector of claim 1, comprising a deletion or modification of US2,US3, US4, US5, US6, US11, or UL97, or a homolog thereof.
 5. Therecombinant RhCMV or HCMV vaccine vector of claim 1, comprising adeletion of Rh158-166 or a homolog thereof.
 6. The recombinant RhCMV orHCMV vaccine vector of claim 1, wherein the RhCMV or HCMV vaccine vectoris a tropism-restricted vector.
 7. The recombinant RhCMV or HCMV vaccinevector of claim 6, wherein the tropism-restrictive vector lacks genesrequired for optimal growth in certain cell types or contains targetsfor tissue-specific micro-RNAs in genes essential for viral replicationor wherein the tropism-restrictive vector has an epithelial, centralnervous system (CNS), or macrophage deficient tropism, or a combinationthereof.
 8. The recombinant RhCMV or HCMV vaccine vector of claim 1,wherein the RhCMV or HCMV vaccine vector has a deletion in a genenon-essential for growth in vivo.
 9. The recombinant RhCMV or HCMVvaccine vector of claim 8, wherein the gene is selected from the groupconsisting of the RL11 family, the pp65 family, the US12 family, and theUS28 family.
 10. The recombinant RhCMV vaccine vector of claim 9,wherein the RhCMV gene is selected from the group consisting ofRh13-Rh29, Rh111-Rh112, Rh191-Rh202, and Rh214-Rh220, or wherein theRhCMV gene is selected from the group consisting of Rh13.1, Rh19, Rh20,Rh23, Rh24, Rh112, Rh190, Rh192, Rh196, Rh198, Rh199, Rh200, Rh201,Rh202, and Rh220.
 11. The recombinant HCMV vaccine vector of claim 9,wherein the HCMV gene region is selected from the group consisting ofRL11, UL6, UL7, UL9, UL11, UL83 (pp65), US12, US13, US14, US17, US18,US19, US20, US21, and UL28.
 12. The recombinant RhCMV or HCMV vaccinevector of claim 1, wherein the vector comprises a deletion in a RhCMV orHCMV gene that is essential for replication within a host, disseminationwithin a host, or spreading from host to host.
 13. The recombinant RhCMVor HCMV vaccine vector of claim 12, wherein the essential gene is UL94,UL32, UL99, UL115, or UL44, or a homolog thereof.
 14. The recombinantRhCMV or HCMV vaccine vector of claim 1, wherein the vector comprises adeletion in gene UL82 or a homolog thereof.
 15. The recombinant RhCMV orHCMV vaccine vector of claim 1, wherein the vector comprises a nucleicacid sequence encoding US2, US3, or US6, or a homolog thereof, whereinthe vector does not encode a functional US11.
 16. The recombinant RhCMVor HCMV vaccine vector of claim 15, wherein the nucleic acid sequenceencodes US2, US3, and US6.
 17. The recombinant RhCMV or HCMV vaccinevector of claim 15, wherein the nucleic acid encoding a US11 openreading frame is deleted.
 18. The recombinant RhCMV or HCMV vaccinevector of claim 15, wherein the vector comprises a nucleic acid sequenceencoding US11, and wherein the nucleic acid sequence encoding US11comprises a point mutation, a frameshift mutation, and/or a deletion ofone or more nucleotides of the nucleic acid sequence encoding US11. 19.The recombinant RhCMV or HCMV vaccine vector of claim 18, wherein thevector lacks the tegument protein pp65.
 20. The recombinant RhCMV orHCMV vaccine vector of claim 1, wherein the vector does not express anactive UL130 protein.
 21. The recombinant RhCMV or HCMV vaccine vectorof claim 1, wherein the RhCMV vaccine vector is Rh68-1 or Rh68-1.2. 22.The recombinant RhCMV or HCMV vaccine vector of claim 1 furthercomprising a microRNA recognition element (MRE) operably linked to a CMVgene that is essential or augmenting for CMV growth, and wherein the MREsilences expression in the presence of a microRNA that is expressed by acell of myeloid lineage.
 23. A pharmaceutical composition comprising therecombinant RhCMV or HCMV vaccine vector of claim 1, and apharmaceutically acceptable carrier.
 24. A method for treatment orprevention of tuberculosis or eliciting an immune response to a Mtbantigen comprising administering to a subject in need thereof at leastone recombinant RhCMV or HCMV vaccine vector of claim
 1. 25. The methodof claim 24, wherein the recombinant RhCMV or HCMV vaccine vector isadministered to the subject intravenously, intramuscularly,intraperitoneally, intranasally, orally, or as an aerosol.
 26. A Mtbantigen, wherein said Mtb antigen is a fusion protein, wherein saidfusion protein comprises the following Mtb proteins or antigenicfragments thereof in the order listed:Ag85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.
 27. The human cytomegalovirus(HCMV) vector of claim 1, comprising a nucleic acid sequence encoding anexpressible Mtb antigen, wherein said nucleic acid sequence encodes theMtb antigen Ag85A-ESAT6-Rv3407-Rv2626c-RpfA-RpfD.
 28. The RhCMV or HCMVvector of claim 1, wherein said nucleic acid sequence encoding an Mtbantigen is a bacterial codon optimized sequence or a mammalian codonoptimized sequence.